Methods and compositions for macrophage polarization

ABSTRACT

Disclosed herein are compositions and methods comprising extracellular vesicles comprising nucleic acid that target genes, leading to macrophage polarization of tumor associated macrophages. In certain embodiments, disclosed herein are methods and compositions for increasing macrophage polarization for the treatment of cancer.

FIELD OF THE INVENTION

This invention relates to compositions and methods comprisingextracellular vesicles harboring nucleic acid that target genes, leadingto macrophage polarization of tumor associated macrophages.

BACKGROUND

Immunotherapy is the treatment of disease by inducing, enhancing, orsuppressing the immune response. Cancer immunotherapy usually has fewerside effects than traditional cancer therapies, such as chemotherapy andradiation therapy. In one approach, cancer immunotherapy has been usedto stimulate the patient's own macrophages to attack cancer cells.Macrophages display different phenotypes, e.g., they can becancer-promoting or they can possess anti-cancer activity. The M2phenotype, or “alternatively activated macrophages,” typically exhibitcancer-promoting activities, such as the suppression of the immunesystem and the production of extracellular matrix- and tissue-remodelingactivities. The M1 phenotype, or “classically activated macrophages,”typically exhibit anti-cancer activities such as the phagocytosis oftumor cells and the stimulation of adaptive immunity so that tumor cellscan be recognized and attacked. Improvements in immunomodulatory methodsand compositions that promote macrophage polarization (i.e., conversionto the M1 phenotype) are needed.

SUMMARY

Aspects of the disclosure encompass an extracellular vesicle comprisingone or more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype. In some aspects the disclosure encompasses an extracellularvesicle that is an exosome comprising one or more immunomodulatingcomponent(s) that, upon contact with a macrophage, selectivelyrepolarizes the macrophage from an M2 to an M1 phenotype

In some aspects, the extracellular vesicle, e.g., the exosome, comprisesone or more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and the immunomodulating component(s) is a nucleic acid,e.g., an inhibitory RNA, e.g., an antisense RNA, an siRNA, an shRNA, amiRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or, e.g., an antisenseoligonucleotide (ASO) or, e.g., the immunomodulating component is anantisense oligonucleotide comprising a sequence at least 95% identicalto a sequence selected from SEQ ID NOs:1-6.

In some aspects, the extracellular vesicle, e.g., the exosome, comprisesone or more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and the immunomodulating (components), e.g., a nucleic acid,e.g., an inhibitory RNA, e.g., an antisense RNA, an siRNA, an shRNA, amiRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or, e.g., an antisenseoligonucleotide (ASO) or, e.g., the immunomodulating component is anantisense oligonucleotide comprising a sequence at least 95% identicalto a sequence selected from SEQ ID NOs:1-6, inhibit(s) at least onemacrophage target gene, e.g., at least one gene is selected from thegroup consisting of: KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300,LKB1, AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase,Glutaminase, CEBP/β, Pi3Kγ, and PKM2, e.g., STAT3, STAT6, CEBP/β, Pi3Kγ,KRAS, and HIF1-alpha, e.g., STAT3, e.g., KRAS.

In some aspects, the extracellular vesicle, e.g., the exosome, comprisesone or more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and the immunomodulating component is an antisenseoligonucleotide that targets STAT3.

In some aspects, the extracellular vesicle, e.g., the exosome, comprisesone or more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and the immunomodulating component is an antisenseoligonucleotide that targets KRAS.

In some aspects, the extracellular vesicle, e.g., the exosome, comprisesone or more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and the immunomodulating component is an inhibitory RNA,e.g., an antisense RNA, an siRNA, an shRNA, a miRNA, a lncRNA, apri-miRNA or a pre-miRNA that targets KRAS, e.g., wild-type human KRAS,or wild-type human KRAS and also mouse KRAS^(G12D).

In any of the above-described aspects, the extracellular vesicle, e.g.,the exosome, comprises one or more immunomodulating component(s), e.g.,a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA,a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact witha macrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and the macrophage is a tumor- (e.g., pancreatic tumor)resident macrophage.

In any of the above-described aspects, the extracellular vesicle, e.g.,the exosome, comprises one or more immunomodulating component(s), e.g.,a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA,a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact witha macrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and further comprise an additional immunomodulatingcomponent, e.g., a small molecule drug, an antibody or active fragmentthereof, e.g., an immune checkpoint inhibitor that binds to CTLA-4,PD-1, or PD-L1, or an inhibitor that binds to CSF1-R, or a therapeuticprotein or active fragment thereof.

In any of the above-described aspects, the extracellular vesicle, e.g.,the exosome, comprises one or more immunomodulating component(s), e.g.,a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA,a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact witha macrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype, and further comprise an additional immunomodulatingcomponent, e.g., an antibody or active fragment thereof, e.g., an immunecheckpoint inhibitor that binds to CTLA-4, PD-1, or PD-L1, or aninhibitor that binds to CSF1-R, wherein the antibody or active fragmentthereof comprises CDRs that are at least 95% identical to the CDRs ofIpilimumab, or at least 95% identical to the CDRs of Nivolumab, or atleast 95% identical to the CDRs of Cemiplimab, or at least 95% identicalto the CDRs of Pembrolizumab, or at least 95% identical to the CDRs ofAtezolizumab, or at least 95% identical to the CDRs of Avelumab, or atleast 95% identical to the CDRs of Durvalumab, or at least 95% identicalto the CDRs of Pexidartinib, or at least 95% identical to the CDRs ofPLX7486, or at least 95% identical to the CDRs of ARRY-382, or at least95% identical to the CDRs of JNJ-40346527, or at least 95% identical tothe CDRs of BLZ945, or at least 95% identical to the CDRs ofEmactuzumab, or at least 95% identical to the CDRs of AMG820, or atleast 95% identical to the CDRs of IMC-CS4, or at least 95% identical tothe CDRs of Cabiralizumab., or wherein the antibody or active fragmentthereof is at least one antibody selected from the group consisting ofIpilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab,Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527,BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab, or wherein theantibody or active fragment thereof is at least one antibody thatcompetes for binding with antibody selected from the group consisting ofIpilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab,Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527,BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab.

In any of the above-described aspects, wherein the extracellularvesicle, e.g., the exosome, comprises one or more immunomodulatingcomponent(s), e.g., a nucleic acid, inhibitory RNA, antisense RNA,siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or anASO that, upon contact with a macrophage, selectively repolarizes themacrophage from an M2 to an M1 phenotype and further comprise anadditional immunomodulating component, e.g., an antibody or activefragment thereof, e.g., an immune checkpoint inhibitor that binds toCTLA-4, PD-1, or PD-L1, or an inhibitor that binds to CSF1-R, whereinthe antibody or active fragment thereof comprises CDRs that are at least95% identical to the CDRs of Ipilimumab, or at least 95% identical tothe CDRs of Nivolumab, or at least 95% identical to the CDRs ofCemiplimab, or at least 95% identical to the CDRs of Pembrolizumab, orat least 95% identical to the CDRs of Atezolizumab, or at least 95%identical to the CDRs of Avelumab, or at least 95% identical to the CDRsof Durvalumab, or at least 95% identical to the CDRs of Pexidartinib, orat least 95% identical to the CDRs of PLX7486, or at least 95% identicalto the CDRs of ARRY-382, or at least 95% identical to the CDRs ofJNJ-40346527, or at least 95% identical to the CDRs of BLZ945, or atleast 95% identical to the CDRs of Emactuzumab, or at least 95%identical to the CDRs of AMG820, or at least 95% identical to the CDRsof IMC-CS4, or at least 95% identical to the CDRs of Cabiralizumab., orwherein the antibody or active fragment thereof is at least one antibodyselected from the group consisting of Ipilimumab, Nivolumab, Cemiplimab,Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib,PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4and Cabiralizumab, or wherein the antibody or active fragment thereof isat least one antibody that competes for binding with antibody selectedfrom the group consisting of Ipilimumab, Nivolumab, Cemiplimab,Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib,PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4and Cabiralizumab, it further comprises PTGFRN or a fragment thereof,and the antibody or fragment thereof may optionally be fused to thePTGFRN or the fragment thereof.

In any of the above-described aspects, the comparison is determinedusing an assay selected from the group consisting of an extracellularvesicle uptake assay, a target gene expression assay, a downstream geneexpression assay, a cytokine release assay and a macrophage cell surfaceprotein assay.

In any of the above-described aspects, the M2 macrophage is a tumorassociated macrophage is a tumor associated macrophage selected from thegroup consisting of a M2a, M2b, and M2c macrophage.

In any of the above-described aspects, the M1 macrophage exhibitsincreased secretion of inflammatory cytokines and chemokines selectedfrom the group consisting of INFγ, IL-12, IL-23, TNFα, IL-6, IL-1,CSCL9, CXCL10 and CXCL11 compared to the M2 macrophage prior topolarization, and/or exhibits decreased secretion of immunosuppressivecytokines and chemokines selected from the group consisting IL-10, TGFβ,PGE2, CCL2, CCL17, CCL18, CCL22 and CCL24 compared to the M2 macrophageprior to polarization, and or expresses increased tumor associatedantigen compared to the M2 macrophage prior to polarization, and/orincreases stimulation of CD8⁺ T-Cells and/or Natural Killer cellscompared to the M2 macrophage prior to polarization.

In aspects, the disclosure encompasses a pharmaceutical compositioncomprising the extracellular vesicle, e.g., exosome, that comprises oneor more immunomodulating component(s) that, upon contact with amacrophage, selectively repolarizes the macrophage from an M2 to an M1phenotype.

In some aspects, the disclosure encompasses a method of treating adisease (e.g., cancer such as, e.g., pancreatic cancer) in a patient(e.g., a human) in need thereof, comprising administering (e.g., via aroute selected from the group consisting of intravenous, intraperitonealand intratumoral administration) the extracellular vesicle of any of theabove-described aspects, e.g., the exosome that comprises one or moreimmunomodulating component(s) (e.g., an inhibitory RNA targeting aproto-oncogene (e.g., KRAS)) that, upon contact with a macrophage,selectively repolarizes the macrophage from an M2 to an M1 phenotype ofany of the above-described aspects, or comprising administering apharmaceutical composition comprising the extracellular vesicle of anyof the above-described aspects, e.g., the exosome that comprises one ormore immunomodulating component(s) that, upon contact with a macrophage,selectively repolarizes the macrophage from an M2 to an M1 phenotype ofany of the above-described aspects.

In some aspects, the disclosure encompasses the methods of treatmentdescribed above and further comprises a second therapy, e.g., a surgicaltherapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy,or immunotherapy.

In some aspects, the disclosure encompasses methods of modulating geneexpression in a macrophage comprising contacting the macrophage with anextracellular vesicle comprising one or more immunomodulating componentsthat inhibit at least one gene and thereby increase macrophagepolarization from an M2 to an M1 phenotype, as compared to contactingthe macrophage with equimolar amount(s) of the immunomodulatingcomponents alone.

In some aspects, the methods of modulating gene expression in amacrophage comprising contacting the macrophage ex vivo or in vitro withan extracellular vesicle comprising one or more immunomodulatingcomponents that inhibit at least one gene and thereby increasemacrophage polarization from an M2 to an M1 phenotype, as compared tocontacting the macrophage with equimolar amount(s) of theimmunomodulating components alone.

In some aspects, the methods of modulating gene expression in amacrophage comprising contacting the macrophage in vivo, with anextracellular vesicle (e.g., by administering the extracellular vesicleto a subject, e.g., a human subject, e.g., by a route selected from thegroup consisting of intravenous, intraperitoneal and intratumoraladministration) comprising one or more immunomodulating components thatinhibit at least one gene and thereby increase macrophage polarizationfrom an M2 to an M1 phenotype, as compared to contacting the macrophagewith equimolar amount(s) of the immunomodulating components alone.

In some aspects, the subject of the above-disclosed methods ofmodulating gene expression in a macrophage, is suffering from acondition selected from cancer (e.g., pancreatic cancer) and fibrosis

In some aspects, the extracellular vesicle used in any of theabove-disclosed methods of modulating gene expression in a macrophage isan exosome, and the immunomodulating component is a nucleic acid, e.g.,an inhibitory RNA, such as, e.g., an antisense RNA, an siRNA, an shRNA,a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA.

In some aspects, the extracellular vesicle used in any of theabove-disclosed methods of modulating gene expression in a macrophage isan exosome, and the immunomodulating component is a nucleic acid, e.g.,an ASO.

In some aspects, the extracellular vesicle used in any of theabove-disclosed methods of modulating gene expression in a macrophage isan exosome, and the immunomodulating component is a nucleic acid, e.g.,an antisense oligonucleotide comprising a sequence at least 95%identical to a sequence selected from SEQ ID NOs: 1-6 or an antisenseoligonucleotide comprising a sequence selected from SEQ ID NOs: 1-6, andthe at least one gene is selected from the group consisting of KRAS,HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6,n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, Glutaminase, CEBP/β,Pi3Kγ, and PKM2, or from the group consisting of: STAT3, STAT6, CEBP/β,Pi3Kγ, KRAS, and HIF1-alpha, or is STAT3, or is KRAS, and if KRAS, theimmunomodulatory component can optionally be an inhibitory RNA thattargets wild-type human KRAS.

In some aspects, the disclosure provides a method of treating pancreaticcancer in a subject comprising: administering to the subject anextracellular vesicle comprising an inhibitory RNA targeting humanwild-type KRAS; wherein the treatment increases the percentage ofpolarization of tumor-resident macrophages from an M2 to an M1 phenotypeto a greater level than that observed in a patient treated with aninhibitory RNA targeting human KRAs^(G12D).

In some aspects the disclosure provides the above-described method oftreating pancreatic cancer in a subject, wherein the percentage ofpolarization of tumor-resident macrophages is determined using anex-vivo assay of tumor-resident macrophages obtained from a tumorsample.

Provided herein are compositions and methods comprising extracellularvesicles selected, enriched, or engineered with one or moreimmunomodulating components that can modify the activity of macrophages,promoting switching of macrophages from the M2 to the M1 phenotype(macrophage polarization) and boosting the patient's immune system tofight cancer.

Accordingly, in a first aspect, provided herein is an extracellularvesicle comprising one or more immunomodulating components, e.g.,nucleic acid molecules that inhibits at least one gene in a target cell.In certain embodiments the target cell is a macrophage and the geneinhibition increases macrophage polarization from the M2 to M1 phenotypeas compared to an equimolar amount of the immunomodulating component(s)alone. In certain embodiments, the extracellular vesicle is an exosome.In certain embodiments, the nucleic acid is an inhibitory RNA. Incertain embodiments, the inhibitory RNA is an antisense RNA, an siRNA,an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA. In certainembodiments, the at least one gene is selected from the group consistingof: KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK,STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase,Glutaminase, CEBP/β, Pi3Kγ, and PKM2. In certain embodiments, the geneis KRAS. In certain embodiments, the nucleic acid is an inhibitory RNAthat targets wild-type human KRAS. In certain embodiments, theinhibitory RNA also targets mouse Kras^(G12D). In certain embodiments,the macrophage is a tumor resident macrophage. In certain embodiments,the tumor is a pancreatic tumor. In certain embodiments, theextracellular vesicle further comprises an additional immunomodulatingcomponent. In certain embodiments, the additional immunomodulatingcomponent is a small molecule drug, an antibody or a therapeuticprotein. In certain embodiments, the antibody is an immune checkpointinhibitor. In certain aspects, provided herein is a pharmaceuticalcomposition comprising any of the above mentioned extracellularvesicles.

In certain aspects, described herein is a method of treating a diseasein a patient in need thereof comprising administering the extracellularvesicles or the pharmaceutical compositions described herein to thepatient, thereby treating the disease in the patient. In certainembodiments, the disease is a cancer. In certain embodiments, the canceris pancreatic cancer. In some embodiments, the disease is a fibroticcondition. In some embodiments, the fibrotic condition is lung fibrosis,liver fibrosis, or pancreatic fibrosis. In certain embodiments, theliver fibrosis is non-alcoholic steatohepatitis, or NASH. In certainembodiments, the patient is human. In certain embodiments, the nucleicacid is an inhibitory RNA targeting a proto-oncogene. In certainembodiments, the proto-oncogene is human KRAS. In certain embodiments,the cancer is pancreatic cancer. In certain embodiments, the methodsfurther comprise performing at least a second therapy. In certainembodiments, the second therapy comprises a surgical therapy,chemotherapy, radiation therapy, cryotherapy, hormonal therapy, orimmunotherapy.

In certain embodiments, the target cell M2 macrophage is a tumorassociated macrophage selected from the group consisting of a M2a, M2b,and M2c macrophage. In certain embodiments, the M1 macrophage exhibitsincreased secretion of inflammatory cytokines and chemokines selectedfrom the group consisting of INFγ, IL-12, IL-23, TNFα, IL-6, IL-1,CSCL9, CXCL10 and CXCL11 compared to the M2 macrophage prior topolarization. In certain embodiments, the M1 macrophage exhibitsdecreased secretion of immunosuppressive cytokines and chemokinesselected from the group consisting IL-10, TGFβ, PGE2, CCL2, CCL17,CCL18, CCL22 and CCL24 compared to the M2 macrophage prior topolarization. In certain embodiments, the M1 macrophage expressesincreased tumor associated antigen compared to the M2 macrophage priorto polarization. In certain embodiments, the M1 macrophage increasesstimulation of CD8⁺ T-Cells and/or Natural Killer cells compared to theM2 macrophage prior to polarization.

In certain aspects, described herein is a method of treating pancreaticcancer in a subject comprising administering to the subject anextracellular vesicle comprising an inhibitory RNA targeting humanwild-type KRAS; wherein the treatment increases the percentage ofpolarization of tumor-resident macrophages from the M2 to M1 phenotypeto a greater level than that observed in a patient treated with aninhibitory RNA targeting human KRAS^(G12D).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gene expression levels of Stat 3, Stat 6, Cebpβ-1,Pi3Kγ, CEBPβ-2, and Kras after transfection of increasing amounts ofsiRNAs targeting each of the gene.

FIG. 2 shows Arg1 expression after transfection of increasing amounts ofsiRNAs targeting Stat 3, Stat 6, Cebpβ-1, Pi3Kγ, CEBPβ-2, and Krasrespectively. Scrambled siRNA transfected macrophages were used ascontrol.

FIG. 3 shows the exosome uptake as determined by total cellular GFPintensity of six macrophage subpopulations (M0, M1, M2a, M2c, M2++, andTAM).

FIG. 4 shows the uptake of free antisense oligos (Free-ASO) andASO-loaded exosomes (Exo-ASO) by M2 or M0 macrophages as measured bytotal fluorescence intensity.

FIG. 5A shows the exosome uptake by macrophages in vivo afterintraperitoneal injection of 1×10¹¹ or 1×10¹² GFP-containing exosomes innaïve mice. FIG. 5B shows the native exosome and Exo-ASO update by tumorcells and macrophages in vivo after injection of a single dose of nativeunlabeled exosomes or Cy5-labeled Exo-ASO in B16F10 tumor-bearing mice.

FIG. 6 shows Stat 3 expression in M2 polarized murine RAW264.7 cellsafter incubation with 5 μM free cholesterol-tagged ASO or either 1×10⁵or 1×10⁶ exosomes loaded with the cholesterol-tagged ASO. Five differentcholesterol-tagged ASOs targeting STAT3 were tested: ASO 4.1,double-stranded MOE chemistry; ASO 5.1 and ASO 5.2, single-strandedO-methyl chemistry; ASO 5.3 and ASO 5.4, single-stranded MOE chemistry.Untreated polarized macrophages and polarized macrophages incubated withunmodified exosomes were used as negative control. Stat 3 siRNAtransfected macrophages were used as positive control.

FIG. 7 shows Arg1 transcript level in M2 polarized murine RAW264.7 cellsafter incubation with 5 μM free cholesterol-tagged ASO or either 1×10⁵or 1×10⁶ exosomes loaded with the cholesterol-tagged ASO. Five differentcholesterol-tagged ASOs targeting STAT3 were tested: ASO 4.1,double-stranded MOE chemistry; ASO 5.1 and ASO 5.2, single-strandedO-methyl chemistry; ASO 5.3 and ASO 5.4, single-stranded MOE chemistry.Untreated polarized macrophages and polarized macrophages incubated withunmodified exosomes were used as negative control. Stat 3 siRNAtransfected macrophages were used as positive control.

FIG. 8A shows STAT3 expression in M2 polarized murine RAW264.7 cellsafter incubation with high (4 μM) dose of ASO or high (4 μM) and low(0.4 μM) doses of Exo-ASO for STAT3. FIG. 8B shows KRAS expression in M2polarized murine RAW264.7 cells after incubation with high (4 μM) highdose of ASO or Exo-ASO for KRAS. FIG. 8C shows C/EBPβ expression in M2polarized murine RAW264.7 cells after incubation with high (4 μM) andlow (0.4 μM) doses of ASO or Exo-ASO for C/EBPβ.

FIG. 9A shows STAT3 transcript level in primary human macrophages fromthree separate donors after treatment with varying doses of free STAT3ASO or STAT3 Exo-ASO. The IC50 of each treatment and the foldimprovement of STAT3 Exo-ASO compared to free STAT3 ASO are presented.FIG. 9B shows CD163 level in primary human macrophages from threeseparate donors after treatment with varying doses of free STAT3 ASO orSTAT3 Exo-ASO. The IC50 of each treatment and fold improvement of STAT3Exo-ASO compared to free STAT3 ASO are presented.

FIG. 10 shows the target gene expression in human M2 macrophages aftertreatment with 4 μM free ASO or Exo-ASO for HIF1α, Pi3Kγ, CEBP/β, STAT6,and STAT3 respectively. Untreated human M2 macrophages and human M2macrophages treated with 4 μM scrambled Exo ASO were used as control.

FIG. 11 shows CD163 expression inhuman M2 macrophages after treatmentwith 4 μM free ASO or Exo-ASO for HIF1α, Pi3Kγ, CEBP/β, STAT6, and STAT3respectively. Untreated human M2 macrophages and human M2 macrophagestreated with 4 μM scrambled Exo ASO were used as control.

FIG. 12A shows the induction of IL-12 after treatment with 4 μM freeSTAT3 ASO, 4 μM STAT3 Exo-ASO, 4 μM scrambled Exo-ASO, native exosomes,or C188-9, a small molecule inhibitor of STAT3 in the presence orabsence of LPS. FIG. 12B shows the induction of IL-23 after treatmentwith 4 μM free STAT3 ASO, 4 μM STAT3 Exo-ASO, 4 μM scrambled Exo-ASO,native exosomes, or C188-9, a small molecule inhibitor of STAT3 in thepresence or absence of LPS.

FIG. 13A shows the STAT3 transcript level after treatment withunmodified exosomes (EV only), and concentration-matched Stat 3 Free ASOand Stat 3 Exo-ASO.

FIG. 13B shows the CD163 transcript level after treatment withunmodified exosomes (EV only), and concentration-matched Stat 3 Free ASOand Stat 3 Exo-ASO. FIG. 13C shows the TGFβ transcript level aftertreatment with unmodified exosomes (EV only), and concentration-matchedStat 3 Free ASO and Stat 3 Exo-ASO. FIG. 13D shows the STAT6 transcriptlevel after treatment with unmodified exosomes (EV only), andconcentration-matched Stat 3 Free ASO and Stat 3 Exo-ASO.

FIG. 14 shows the TGFβ expression level after treatment with exosomesloaded with ASOs against STAT3 (both MOE and LNA chemistry), STAT6 (MOEchemistry), and CEBP/β (MOE chemistry) respectively.

DETAILED DESCRIPTION

Macrophage polarization is a process by which macrophages adoptdifferent functional programs in response to the signals from theirmicroenvironment. This ability is connected to their multiple roles inthe organism: they are powerful effector cells of the innate immunesystem, but also important in removal of cellular debris, embryonicdevelopment and tissue repair.

Macrophage phenotypes are broadly divided into 2 groups: M1(classically-activated macrophages) and M2 (alternatively-activatedmacrophages). This broad classification is based on in vitro studies, inwhich cultured macrophages were treated with molecules that stimulatedtheir phenotype switching to particular state. M1 macrophages arepro-inflammatory, important in direct host-defense against pathogen,such as phagocytosis and secretion of pro-inflammatory cytokines andmicrobicidal molecules. M2 macrophages have quite the opposite function:regulation of the resolution phase of inflammation and the repair ofdamaged tissues. See, e.g., Wynn, T. A., Chawla, A., & Pollard, J. W.(2013). Origins and Hallmarks of Macrophages: Development, Homeostasis,and Disease. Nature, 496(7446), 445-455; Mills, C. D., Kincaid, K., Alt,J. M., Heilman, M. J., & Hill, A. M. (2000). M-1/M-2 Macrophages and theTh1/Th2 Paradigm. The Journal of Immunology, 164(12), 6166-6173. Manysolid tumors are characterized by a myeloid-rich cellular infiltrate,often comprising a type of M2 macrophage known as tumor-associatedmacrophages (TAMs). M2 macrophages (such as TAMs) express high levels ofphosphorylated STAT3 and STAT6, which promote the expression of themetabolic enzyme Arginase (Arg1). TAMs mediate a number oftumor-promoting activities such as, e.g., promotion of cancer cellmotility, metastasis formation and angiogenesis and TAM formation isdependent on microenvironmental factors which are present in developingtumor. TAMs produce immunosuppressive cytokines such as, e.g., IL-10,TGFβ, PGE2 and a very small amount of NO or ROI and low levels ofinflammatory cytokines (IL-12, IL-1β, TNFα, IL-6). As compared to“classically-activated” M1 macrophages, presentation of tumor-associatedantigens by TAMs is decreased, as is stimulation of the anti-tumorfunctions of T and NK cells. Unlike M1 macrophages, TAMs are unable tolyse tumor cells.https://en.wikipedia.org/wiki/Macrophage_polarization-cite_note-Sica2008-31Thus, targeting of TAMs and other M2 macrophages provides a noveltherapeutic strategy against cancer, as has been demonstrated throughthe delivery of agents to either alter the recruitment and distributionof TAMs, deplete existing TAMs, or induce the re-education of TAMs froman M2 to an M1 phenotype.

The exosome therapeutics of the present invention have selective effectson M2 macrophages to promote a tissue-resident microenvironment to treatdiseases like cancer and fibrosis by “repolarizing” the aberrantmacrophages to an M1 phenotype in the context of these disease. Theexosomes described herein are precisely engineered with variousbiologically active molecules onto the exosome surface or inside theexosome lumen to create candidates that engage pathways that repolarizethe M2 macrophages to M1 phenotype to treat human disease.

Tumor associated M2 polarized macrophages, or TAMs, can effectivelysuppress T cell proliferation and effector function and promote tumorgrowth. Reversion of TAMs back to an M1 phenotype has also been reportedusing an antibody which depletes macrophages directed against the CSF-1receptor. These approaches have shown limited success in clinic trialsdue to a narrow therapeutic window and lack of specificity due totargeting all macrophages and not just the aberrant M2 macrophages asintended. Such wholesale depletion of macrophages would be expected toresult in increased infection risk and other safety concerns. Theexosomes of the present invention more selectively target the M2macrophages due to natural exosome tropism for macrophages and tip thebalance of function towards the desired M1 phenotype. These exosomesrepolarize macrophages, resulting in the production of the desiredspectrum of inflammatory cytokines needed for anti-tumor immuneresponses. As shown in the examples below, the exosomes havereprogrammed the immunosuppressive M2 macrophages to M1 phenotype invitro.

In addition to the immune suppression induced in the tumormicroenvironment, M2 polarized macrophages secrete large amounts oftransforming growth factor beta, or TGFβ, an important cytokine involvedin cellular signaling, which induces fibroblast accumulation leading tocollagen deposition and tissue remodeling, ultimately resulting intissue fibrosis. Reprogramming these M2 macrophages to reduce TGFβproduction by targeting key signaling molecules, like STAT3, has beenshown to have beneficial activity in preclinical models of lungfibrosis. Several small molecule approaches to targeting STAT3 have beenemployed, but the lack of specificity of inhibition only within theaberrant M2 macrophages has prevented this approach to treatment frombeing viable. Certain exosomes described here selectively target keypathways in M2 macrophages allowing them to target STAT3 and othercellular pathways in lung and other tissue fibrosis syndromes.

Disclosed herein are extracellular vesicles useful for modulatingmacrophages of the immune system. These extracellular vesicles compriseone or more immunomodulating component(s) that, upon contact with amacrophage, inhibit at least one macrophage target gene therebyincreasing macrophage polarization from an M2 to an M1 phenotype, ascompared to equimolar amount(s) of the one or more immunomodulatingcomponent(s) alone. Also provided are methods for producing theextracellular vesicles, and methods of using these extracellularvesicles to treat cancer and other immune system related diseases suchas, e.g., fibrotic conditions.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such can, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges can independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

It is noted that, as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Similarly, the term “at least one”includes plural referents (i.e., is equivalent to the phrase “one ormore,” unless the context clearly dictates otherwise). It is furthernoted that the claims can be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a negativelimitation.

The term “about” when used to modify numeric values, is intended toencompass variations in the stated values that are functionallyequivalent to the stated values for purposes of practicing the describedtechnology, as can be readily determined by the skilled artisan. Incertain embodiments the term “about” includes +/−5%, +/−10%, +/−20%,+/−30%, +/−40% or +/−50% variation from the stated values.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing the subject invention, subject systems for use inpracticing the subject methods will be discussed in greater detail,followed by a review of associated methods.

As used herein, the term “extracellular vesicle” refers to acell-derived vesicle comprising a membrane that encloses an internalspace. Extracellular vesicles comprise all membrane-bound vesicles thathave a smaller diameter than the cell from which they are derived.Generally extracellular vesicles range in diameter from 20 nm to 1000nm, and can comprise various macromolecular cargo either within theinternal space, displayed on the external surface of the extracellularvesicle, and/or spanning the membrane. The cargo can comprise nucleicacids, proteins, carbohydrates, lipids, small molecules, and/orcombinations thereof. By way of example and without limitation,extracellular vesicles include apoptotic bodies, fragments of cells,vesicles derived from cells by direct or indirect manipulation (e.g., byserial extrusion or treatment with alkaline solutions), vesiculatedorganelles, and vesicles produced by living cells (e.g., by directplasma membrane budding or fusion of the late endosome with the plasmamembrane). Extracellular vesicles can be derived from a living or deadorganism, explanted tissues or organs, and/or cultured cells. Species ofextracellular vesicles include exosomes and nanovesicles, as described,e.g., in co-owned U.S. Pat. No. 10,195,290, incorporated herein byreference for all purposes.

As used herein the term “exosome” refers to a cell-derived small(between 20-300 nm in diameter, more preferably 40-200 nm in diameter)vesicle comprising a membrane that encloses an internal space, and whichis generated from the cell by direct plasma membrane budding or byfusion of the late endosome with the plasma membrane. The exosome is aspecies of extracellular vesicle. The exosome comprises lipid or fattyacid and polypeptide and optionally comprises a payload (e.g., atherapeutic agent), a receiver (e.g., a targeting moiety), apolynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., asimple sugar, polysaccharide, or glycan) or other molecules. The exosomecan be derived from a producer cell, and isolated from the producer cellbased on its size, density, biochemical parameters, or a combinationthereof.

As used herein, the term “nanovesicle” refers to a cell-derived small(between 20-250 nm in diameter, more preferably 30-150 nm in diameter)vesicle comprising a membrane that encloses an internal space, and whichis generated from the cell by direct or indirect manipulation such thatthe nanovesicle would not be produced by the producer cell without themanipulation. Appropriate manipulations of the producer cell include butare not limited to serial extrusion, treatment with alkaline solutions,sonication, or combinations thereof. The production of nanovesicles can,in some instances, result in the destruction of the producer cell.Preferably, populations of nanovesicles are substantially free ofvesicles that are derived from producer cells by way of direct buddingfrom the plasma membrane or fusion of the late endosome with the plasmamembrane. The nanovesicle is a species of extracellular vesicle. Thenanovesicle comprises lipid or fatty acid and polypeptide, andoptionally comprises a payload (e.g., a therapeutic agent), a receiver(e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA,or DNA), a sugar (e.g., a simple sugar, polysaccharide, or glycan) orother molecules. The nanovesicle, once it is derived from a producercell according to the manipulation, can be isolated from the producercell based on its size, density, biochemical parameters, or acombination thereof.

The term “M2 phenotype” as used herein, refers to macrophages thatexhibit one or more tumor promoting activities or express markers knownin the art to be associated with the M2 phenotype such as, but notlimited to, reduced or lack of stimulation of CD8⁺ T-Cells and/orNatural Killer cells; lack of phagocytosis of tumor cells; secretionand/or expression of M2 associated cytokines (e.g., IL-10, TGFβ, PGE2,CCL2, CCL17, CCL18, CCL22 and CCL24); secretion and/or expression ofgrowth factors (e.g., VEGF-A, VEGF-C, EGF, and TGF-β); secretion and/orexpression of metastatic enzymes (e.g., matrix metalloproteinases MMP2,MMP9, cysteine cathepsin proteases); secretion/expression ofimmunosuppressive factors (e.g., Arginase I (ArgI), which withdraws thesubstrate L-arginine from inducible nitric oxide synthase (iNOS));expression of cell surface markers YM1, FIZZ1, Dectin-1, MGL, expressionof M2 associated miRNAs (e.g., miRNA146a, miRNA let 7b, and miR-223)(see, e.g., Mosser, D. M., & Edwards, J. P. (2008). Exploring the fullspectrum of macrophage activation. Nature Reviews Immunology, 8(12),958-96; Murray, P. J., Allen, J. E., Biswas, S. K., Fisher, E. A.,Gilroy, D. W., Goerdt, S., Wynn, T. A. (2014). Macrophage activation andpolarization: nomenclature and experimental guidelines. Immunity, 41(1),14-20; and Liu, Y. C., Zou, X. B., Chai, Y. F., & Yao, Y. M. (2014).Macrophage polarization in inflammatory diseases. International Journalof Biological Sciences, 10(5), 520-5299, and references cited therein,each incorporated by reference for all purposes) and/or reducedexpression/secretion of M1 associated factors or reduced M1 associatedactivities listed below compared to at least one reference sample,wherein the reference sample comprises a population of M1-activatedmacrophages. M1-activation in vitro is evoked by treatment with TLRligands such as bacterial lipopolysaccharide (LPS)—typical forGram-negative bacteria and lipoteichoic acid (LTA)—typical forGram-positive bacteria, granulocyte-macrophage colony-stimulating factor(GM-CSF) or combination of LPS and interferon-gamma (IFN-γ). See Mills,C. D., Kincaid, K., Alt, J. M., Heilman, M. J., & Hill, A. M. (2000).M-1/M-2 Macrophages and the Th1/Th2 Paradigm. The Journal of Immunology,164(12), 6166-6173; Krausgruber, Thomas, et al. “IRF5 promotesinflammatory macrophage polarization and TH1-TH17 responses.” Natureimmunology 12.3 (2011): 231-238 and Martinez, F. O., & Gordon, S.(2014). The M1 and M2 paradigm of macrophage activation: time forreassessment. F1000Prime Reports, 6(March), 1-13, and references citedtherein, each incorporated herein by reference for all purposes.

The term “M1 phenotype” as used herein, refers to macrophages thatexhibit anti-tumor activities or markers known in the art to beassociated with the M1 phenotype such as, but not limited to,stimulation of CD8⁺ T-Cells and/or Natural Killer cells, phagocytosis oftumor cells, secretion and/or expression of M1 associated cytokines(e.g., INFγ, IL-12, IL-23, TNFα, IL-6, IL-1, CCL5, CSCL9, CXCL10 andCXCL11), expression of M1 associated miRNAs (e.g., miRNA155, miR-33)(see, e.g., Mosser, D. M., & Edwards, J. P. (2008). Exploring the fullspectrum of macrophage activation. Nature Reviews Immunology, 8(12),958-96; Murray, P. J., Allen, J. E., Biswas, S. K., Fisher, E. A.,Gilroy, D. W., Goerdt, S., Wynn, T. A. (2014). Macrophage activation andpolarization: nomenclature and experimental guidelines. Immunity, 41(1),14-20; and Liu, Y. C., Zou, X. B., Chai, Y. F., & Yao, Y. M. (2014).Macrophage polarization in inflammatory diseases. International Journalof Biological Sciences, 10(5), 520-5299, and references cited therein,each incorporated by reference for all purposes) and/or reducedexpression/secretion of M2 associated factors or reduced M2 associatedactivities listed above compared to at least one reference sample,wherein the reference sample comprises a population of M2-activatedmacrophages. M2-activation in vitro is evoked by treatment with IL-4 andIL-13 (see, e.g., Liu, Y. C., Zou, X. B., Chai, Y. F., & Yao, Y. M.(2014). Macrophage polarization in inflammatory diseases. InternationalJournal of Biological Sciences, 10(5), 520-529, incorporated byreferences for all purposes).

The term “macrophage polarization” as used herein, refers to change of amacrophage from an M2 to an M1 phenotype and/or refers to an increase inthe percentage of a population of macrophages found in a patient (e.g.,macrophages associated with a tumor, or circulating macrophages) ofmacrophages exhibiting the M1 phenotype as compared to at least onereference sample (e.g., a sample taken from the same patient prior tothe test sample or historical data). See, e.g., Mills, C. D., Kincaid,K., Alt, J. M., Heilman, M. J., & Hill, A. M. (2000). M-1/M-2Macrophages and the Th1/Th2 Paradigm. The Journal of Immunology,164(12), 6166-6173; Mosser, D. M., & Edwards, J. P. (2008). Exploringthe full spectrum of macrophage activation. Nature Reviews Immunology,8(12), 958-969; and Xue, J., Schmidt, S. V. and Schultze, J. L. (2014),Transcriptome-Based Network Analysis Reveals a Spectrum Model of HumanMacrophage Activation. Immunity, 40(2)L 274-288, each incorporated byreference for all purposes.

The term “extracellular vesicle delivery” or “delivery of extracellularvesicles” refers to the administration and localization of extracellularvesicles to target tissues, cells, and/or organs of the subject. In someembodiments, the immunomodulating component can be delivered to thecytoplasm of a target cell. In other embodiments, the immunomodulatingcomponent is delivered to the membrane of the target cell. In someembodiments, the membrane of the extracellular vesicle fuses with amembrane of a target cell.

As used herein, the term “producer cell” refers to any cell from whichan extracellular vesicle can be isolated. A producer cell is a cellwhich serves as a source for the extracellular vesicle. A producer cellcan share a protein, lipid, sugar, or nucleic acid component with theextracellular vesicle. In some embodiments, the producer cell is amodified or synthetic cell. In some embodiments, the producer cell is acultured or isolated cell. In certain embodiments, the producer cell isa cell line. In some embodiments, the producer cell line is a humanembryonic kidney cell line. In some embodiments, the producer cell lineis a HEK293SF cell line. In certain other embodiments, the producer cellis a primary cell. In some particular embodiments, the producer cell isan immune cell, such as, e.g., a B lymphocyte, a T lymphocyte, adendritic cell, a mast cell, a macrophage, a natural killer cell (NKcell), an antigen presenting cell, a T helper cell, or a regulatory Tcell (Treg cell).

“Membrane” as used herein is a boundary layer that separates an interiorspace from an exterior space comprising one or more biologicalcompounds, typically lipids, and optionally polypeptides and/orcarbohydrates. In some embodiments, the membrane comprises lipids andfatty acids. In some embodiments, the membrane comprises phospholipids,glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols,cholesterols, and phosphatidylserines. In some of these embodiments, themembrane further comprises one or more polypeptide and/or one or morepolysaccharide, such as glycan. The extracellular vesicle comprises amembrane as defined herein.

As used herein, the term “immunomodulating component” refers to atherapeutic agent that acts on a target (e.g., a target gene, includingby way of example but not limitation KRAS, HRAS, NRAS, HIF1-alpha,HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1,A2AB, IDO, TDO, Arginase, Glutaminase, CEBP/β, Pi3Kγ, and PKM2) that iscontacted with the agent, and modifies an immune cell (e.g., amacrophage or other immune cells). The immunomodulating component thatcan be introduced into an extracellular vesicle and/or a producer cellinclude therapeutic agents such as, a polynucleotide, such as aninhibitory nucleic acid (e.g., antisense oligonucleotide (ASO), siRNA,miRNA, antisense RNA, shRNA, lncRNA, pri-miRNA and pre-miRNA), anagonist, an antagonist, an antibody, and/or an antigen-binding fragment,modulators of immune checkpoint inhibitors or ligands of immunecheckpoint inhibitors, surface antigens and derivatives thereof, and/orcytokines and derivatives thereof. In certain embodiments theimmunomodulating component is an inhibitory nucleic acid (e.g.,antisense oligonucleotide (ASO), siRNA, miRNA, antisense RNA, shRNA,lncRNA, pri-miRNA and pre-miRNA). See, e.g., Weiss, B. (ed.): AntisenseOligodeoxynucleotides and Antisense RNA: Novel Pharmacological andTherapeutic Agents, CRC Press, Boca Raton, Fla., 1997; Elbashir S,Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). “Duplexesof 21-nucleotide RNAs mediate RNA interference in cultured mammaliancells”. Nature. 411 (6836): 494-988; Bartel D P (January 2004).“MicroRNAs: genomics, biogenesis, mechanism, and function”. Cell. 116(2): 281-97; Paddison, P J; Caudy, A A; Bernstein, E; Hannon, G J;Conklin, D S (15 Apr. 2002). “Short hairpin RNAs (shRNAs) inducesequence-specific silencing in mammalian cells”. Genes & Development. 16(8): 948-58; Ma L, Bajic V B, Zhang Z (June 2013). “On theclassification of long non-coding RNAs”. RNA Biology. 10 (6): 925-33;and Ambros V, Bartel B, Bartel D P, Burge C B, Carrington J C, Chen X,Dreyfuss G, Eddy S R, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G,Tuschl T (March 2003). “A uniform system for microRNA annotation”. RNA.9 (3): 277-9, each of which is incorporated herein for all purposes.

As used herein, the phrase “nucleic acid molecule that inhibits” or“inhibitory nucleic acid” refers to any nucleic acid that, whenintroduced into a cell so that it interacts with a target gene, resultsin inhibition of the expression or activity of that target gene. Anucleic acid molecule that inhibits, i.e., an inhibitory nucleic acidmay be DNA, or an inhibitory RNA (e.g., siRNA, miRNA, antisense RNA,shRNA, lncRNA, pre-miRNA, or mRNA), wherein said RNA is single stranded,double stranded, or contains both single stranded and double strandedregions. In some embodiments, an inhibitory nucleic acid is an antisenseoligonucleotide (ASO). The ASO can be a single-stranded ordouble-stranded DNA, RNA, or DNA/RNA hybrid. See, e.g., AntisenseOligodeoxynucleotides and Antisense RNA: Novel Pharmacological andTherapeutic Agents, CRC Press, Boca Raton, Fla., 1997, incorporatedherein by reference for all purposes. An “EXO ASO” is an ASO that isphysically associated with an extracellular vesicle such as, e.g., anexosome, through interactions with the vesicle membrane (e.g., as occurswith cholesterol- or fatty-acyl-derivatized ASOs), or by loading intothe vesicle lumen using techniques such as electroporation, or viagenetic engineering of a producer cell such as by, e.g., transfection ortransduction to introduce into the producer cell a construct thatencodes the desired ASO, followed by isolation of extracellular vesiclesfrom the engineered producer cell.

The term “receiver” refers to a molecule that directs the extracellularvesicle to a target and/or promotes the interaction of the extracellularvesicle with the target in the subject. In some embodiments, thereceiver is a polypeptide, also sometimes referred to herein as a“receiver polypeptide.” In some embodiments, the receiver is capable ofincreasing the concentration of the immunomodulating component in thetissue of the subject, such as by directed trafficking to the targettissue of the subject. Examples of receivers include, but are notlimited to, examples listed in Table 3.

The term “target” can refer to a gene, the activity of which is to bemodulated by an immunomodulatory component of the present disclosure(i.e., a target gene). In certain embodiments, the target gene isinhibited by a nucleic acid molecule associated with (i.e., bound to themembrane surface, intercalated within the lipid bilayer, or encapsulatedwithin the vesicle's enclosed volume) extracellular vesicles, such as,e.g., an inhibitory ASO. Examples of target genes include, but are notlimited to, KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1,AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase,Glutaminase, CEBP/β, Pi3Kγ, and PKM2. “Target” can also refer to a cellto which the extracellular vesicles of the present disclosure aredirected (i.e., a target cell). In certain embodiments, the target cellis an immune cell (e.g., a macrophage). In certain embodiments, thetarget cell is a hematopoietic stem cell or pluripotent stem cell. Incertain embodiments, the target cell is a circulating macrophage. Incertain embodiments, the target cell is a tumor resident macrophage(i.e., a macrophage located in the tumor microenvironment, in the tumortissue or near the tumor surface). Additionally, “target” can also referto a protein on a target cell, whose activity is modulated by contactwith an immunomodulatory component of the present disclosure, or aprotein that acts as a ligand for binding the extracellular vesicles ofthe present disclosure (i.e., a target protein), Thus, in certainembodiments, the target protein is on the target cell and interacts withthe extracellular vesicle.

A “therapeutic agent” or “therapeutic molecule” includes a compound ormolecule that, when present in an effective amount, produces a desiredtherapeutic effect, pharmacologic and/or physiologic effect on a subjectin need thereof. It includes any compound, e.g., a small molecule drug,or a biologic (e.g., a polypeptide drug or a nucleic acid drug) thatwhen administered to a subject has a measurable or conveyable effect onthe subject, e.g., it alleviates or decreases a symptom of a disease,disorder or condition.

The term “immune checkpoint inhibitor” or “checkpoint inhibitor” as usedherein, refers to a therapeutic agent that stimulates immune cellactivity by reducing immunosuppressive checkpoint pathways that suppressimmune cells (e.g., agents that inhibit PD-1/PD-L1 (such as NivolumabCemiplimab and Pembrolizumab targeting PD-1, and Atezolizumab, Avelumab,Durvalumab, each targeting PD-L1) and CTLA-4/B7-1/B7-2, such asIpilimumab). See, e.g., Pardoll D M (March 2012). “The blockade ofimmune checkpoints in cancer immunotherapy”. Nature Reviews. Cancer. 12(4): 252-64.

As used herein, the term “antibody” encompasses an immunoglobulinwhether natural or partly or wholly synthetically produced, andfragments thereof. The term also covers any protein having a bindingdomain that is homologous to an immunoglobulin binding domain.“Antibody” further includes a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. Use of the term antibody is meant to includewhole antibodies, polyclonal, monoclonal and recombinant antibodies,fragments thereof, and further includes single-chain antibodies,humanized antibodies, murine antibodies, chimeric, mouse-human,mouse-primate, primate-human monoclonal antibodies, anti-idiotypeantibodies, antibody fragments, such as, e.g., scFv, (scFv)₂, Fab, Fab′,and F(ab′)₂, F(ab1)₂, Fv, dAb, and Fd fragments, diabodies, andantibody-related polypeptides. Antibody includes bispecific antibodiesand multispecific antibodies so long as they exhibit the desiredbiological activity or function. Exemplary antibody compositions includeantibodies that inhibit CTLA-4, such as, e.g., Ipilimumab, those thatinhibit PD-1, such as, e.g., Nivolumab Cemiplimab and Pembrolizumab,those that inhibit PD-L1, such as, e.g., Atezolizumab, Avelumab,Durvalumab, and those that inhibit CSFRI, such as, e.g., Pexidartinib,PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4and Cabiralizumab. The term “antigen-binding fragment” used hereinrefers to fragments of an intact immunoglobulin, and any part of apolypeptide including antigen binding regions having the ability tospecifically bind to the antigen. For example, the antigen-bindingfragment can be a F(ab′)₂ fragment, a Fab′ fragment, a Fab fragment, aFv fragment, or a scFv fragment, but is not limited thereto. A Fabfragment has one antigen binding site and contains the variable regionsof a light chain and a heavy chain, the constant region of the lightchain, and the first constant region CH1 of the heavy chain. A Fab′fragment differs from a Fab fragment in that the Fab′ fragmentadditionally includes the hinge region of the heavy chain, including atleast one cysteine residue at the C-terminal of the heavy chain CH1region. The F(ab′)₂ fragment is produced whereby cysteine residues ofthe Fab′ fragment are joined by a disulfide bond at the hinge region. AnFv fragment is the minimal antibody fragment having only heavy chainvariable regions and light chain variable regions, and a recombinanttechnique for producing the Fv fragment is well-known in the art.Two-chain Fv fragments can have a structure in which heavy chainvariable regions are linked to light chain variable regions by anon-covalent bond. Single-chain Fv (scFv) fragments generally can have adimer structure as in the two-chain Fv fragments in which heavy chainvariable regions are covalently bound to light chain variable regionsvia a peptide linker or heavy and light chain variable regions aredirectly linked to each other at the C-terminal thereof. Theantigen-binding fragment can be obtained using a protease (for example,a whole antibody is digested with papain to obtain Fab fragments, and isdigested with pepsin to obtain F(ab′)₂ fragments), and can be preparedby a genetic recombinant technique. A dAb fragment consists of a VHdomain. Single-chain antibody molecules can comprise a polymer with anumber of individual molecules, for example, dimer, trimer or otherpolymers.

The phrase “nucleic acid molecule” refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases. It includeschromosomal DNA and self-replicating plasmids, vectors, mRNA, tRNA,siRNA, miRNA, etc. The nucleic acid molecule can be recombinant andexogenous polypeptides can be expressed when the nucleic acid isintroduced into a cell. The term encompasses chemically modified nucleicacids, such as those described in, e.g., Selvam C, Mutisya D, Prakash S,Ranganna K, Thilagavathi R. “Therapeutic potential of chemicallymodified siRNA: Recent trends,” Chem Biol Drug Des. 2017 November;90(5):665-678, Locked Nucleic Acids (LNAs) as described in, e.g.,Petersen M, Wengel J (February 2003). “LNA: a versatile tool fortherapeutics and genomics”. Trends Biotechnol. 21 (2): 74-81; and othertypes of clinically-relevant, chemically modified nucleic acids such asthose described in, e.g., Summerton, J; Weller, D (1997). “MorpholinoAntisense Oligomers: Design, Preparation and Properties”. Antisense &Nucleic Acid Drug Development. 7 (3): 187-195; Goodchild, J (2011).Therapeutic oligonucleotides. Methods in Molecular Biology. 764. pp.1-15, each incorporated herein by reference for all purposes.

The term “agonist” refers to a molecule that binds to a receptor andactivates the receptor to produce a biological response. Receptors canbe activated by either an endogenous or an exogenous agonist.Non-limiting examples of endogenous agonist include hormones andneurotransmitters. Non-limiting examples of exogenous agonists includevarious classes of compounds including small molecules, antibodies,synthetic peptides, etc. The agonist can be a full, partial, or inverseagonist.

The term “antagonist” refers to a molecule that blocks or dampens anagonist mediated response rather than provoking a biological responseitself upon bind to a receptor. Many antagonists achieve their potencyby competing with endogenous ligands or substrates at structurallydefined binding sites on the receptors. Non-limiting examples ofantagonists include alpha blockers, beta-blocker, and calcium channelblockers. The antagonist can be a competitive, non-competitive, oruncompetitive antagonist.

As used herein, the term “pharmaceutical composition” refers to one ormore of the compounds described herein, such as, e.g., an extracellularvesicle mixed or intermingled with, or suspended in one or more otherchemical components, such as pharmaceutically-acceptable carriers andexcipients. One purpose of a pharmaceutical composition is to facilitateadministration of preparations of extracellular vesicles to a subject.The term “pharmaceutically-acceptable” and grammatical variationsthereof, refers to compositions, carriers, diluents and reagents capableof administration to or upon a subject without the production ofundesirable physiological effects to a degree that prohibitsadministration of the composition. The term “excipient” or “carrier”refers to an inert substance added to a pharmaceutical composition tofurther facilitate administration of a compound. The term“pharmaceutically-acceptable carrier” or “pharmaceutically-acceptableexcipient” encompasses any of the agents approved by a regulatory agencyof the US Federal government or listed in the US Pharmacopeia for use inanimals, including humans, as well as any carrier or diluent that doesnot cause significant irritation to a subject and does not abrogate thebiological activity and properties of the administered compound.Included are excipients and carriers that are useful in preparing apharmaceutical composition and are generally safe, non-toxic, anddesirable.

As used herein, the terms “isolate,” “isolated,” and “isolating” or“purify,” “purified,” and “purifying” as well as “extracted” and“extracting” are used interchangeably and refer to the state of apreparation (e.g., a plurality of known or unknown amount and/orconcentration) of desired extracellular vesicles, that have undergoneone or more processes of purification, e.g., a selection or anenrichment of the desired extracellular vesicle preparation. In someembodiments, isolating or purifying as used herein is the process ofremoving, partially removing (e.g. a fraction) of the extracellularvesicles from a sample containing producer cells. In some embodiments,an isolated extracellular vesicle composition has no detectableundesired activity or, alternatively, the level or amount of theundesired activity is at or below an acceptable level or amount. Inother embodiments, an isolated extracellular vesicle composition has anamount and/or concentration of desired extracellular vesicles at orabove an acceptable amount and/or concentration. In other embodiments,the isolated extracellular vesicle composition is enriched as comparedto the starting material (e.g. producer cell preparations) from whichthe composition is obtained. This enrichment can be by 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%,99.999%, 99.9999%, or greater than 99.9999% as compared to the startingmaterial. In some embodiments, isolated extracellular vesiclepreparations are substantially free of residual biological products. Insome embodiments, the isolated extracellular vesicle preparations are100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of anycontaminating biological matter. Residual biological products caninclude abiotic materials (including chemicals) or unwanted nucleicacids, proteins, lipids, or metabolites. Substantially free of residualbiological products can also mean that the extracellular vesiclecomposition contains no detectable producer cells and that onlyextracellular vesicles are detectable.

The terms “administration,” “administering” and variants thereof referto introducing a composition, such as an extracellular vesicle, or agentinto a subject and includes concurrent and sequential introduction ofone or more additional compositions or agents on any schedule consistentwith producing a therapeutic effect. Timing of dose administration canbe selected to achieve constant levels within a subject (e.g., plasmalevels) of the administered agent, or to episodic exposure, e.g., toreduce toxicity or prevent desensitization. Methods of achieving theseresults are well known to the ordinarily-skilled practitioner, based onknown pharmacokinetic principles as set out in, e.g., Goodman & Gilman'sThe Pharmacological Basis of Therapeutics (Macmillan Publishing Co.13^(th) Ed.) The introduction of a composition or agent into a subjectis by any suitable route, including orally, pulmonarily, intranasally,parenterally (intravenously, intra-arterially, intramuscularly,intraperitoneally, or subcutaneously), rectally, intralymphatically,intrathecally, periocularly, intra-tumorally or topically.Administration includes self-administration and the administration byanother. A suitable route of administration allows the composition orthe agent to perform its intended function. For example, if a suitableroute is intravenous, the composition is administered by introducing thecomposition or agent into a vein of the subject.

As used herein, the term “modulate,” “modulating,” “modify,” and/or“modulator” generally refers to the ability to alter, by increase ordecrease, e.g., directly or indirectlypromoting/stimulating/up-regulating or interferingwith/inhibiting/down-regulating a specific concentration, level,expression, function or behavior, such as, e.g., to act as an antagonistor agonist. In some instances a modulator can increase and/or decrease acertain concentration, level, activity or function relative to acontrol, or relative to the average level of activity that wouldgenerally be expected or relative to a control level of activity. Theterms “inhibition,” “repression,” “modulation,” are used herein todescribe the changes in the expression or activity of the target gene ascompared to the conditions without the immunomodulating component(s).Inhibition refers to elimination or substantial elimination of the geneexpression or activity. Repression means a reduction, but not completeelimination or substantial elimination, of the gene expression oractivity. Modulation means any alteration, up or down, of the geneexpression or activity.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to modulate a condition inthe subject.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

Percent “identity” between a nucleotide sequence and a referencesequence, is defined as the percentage of single nucleotides in thenucleotide sequence that are identical to the single nucleotides in thereference sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleotide sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA,or MUSCLE software. Those skilled in the art can determine appropriateparameters for aligning sequences, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For further details about the BLAST algorithm, see Mount, D.W. (2004). Bioinformatics: Sequence and Genome Analysis (2nd ed.). ColdSpring Harbor Press. ISBN 978-0-87969-712-9.

Percent “identity” between a polypeptide sequence and a referencesequence, is defined as the percentage of amino acid residues in thepolypeptide sequence that are identical to the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA,or MUSCLE software. Those skilled in the art can determine appropriateparameters for aligning sequences, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For further details about the BLAST algorithm, see Mount, D.W. (2004). Bioinformatics: Sequence and Genome Analysis (2nd ed.). ColdSpring Harbor Press. ISBN 978-0-87969-712-9.

As used herein, the term “substantially” or “substantial” refers, e.g.,to the presence, level, or concentration of an entity in a particularspace, the effect of one entity on another entity, or the effect of atreatment. For example, an activity, level or concentration of an entityis substantially increased if the increase is 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold relative to a baseline.An activity, level or concentration of an entity is also substantiallyincreased if the increase is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, or 500% relative to a baseline.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humanmammals.

Abbreviations used in this application include the following: “mRNA”refers to messenger RNA; “miRNA” refers to microRNA; “siRNA” refers tosmall interfering RNA; “antisense RNA” refers to single stranded RNAthat is complementary to an mRNA, which may additionally comprise DNAnucleotides, and is often referred to as an antisense oligonucleotide or“ASO”; “shRNA” refers to small or short hairpin RNA; “lncRNA” refers tolong non-coding RNA; and “dsDNA” refers to double stranded DNA.

Compositions

Aspects of the subject disclosure include compositions capable ofregulating the immune system. The composition comprises an extracellularvesicle comprising a cell membrane, and at least one immunomodulatingcomponent associated with the cell membrane or enclosed within themembrane-bound enclosed volume. Enclosure within the membrane-boundvolume can be accomplished using techniques including electroporation,lyophilization, or through engineering of producer cells (such as, e.g.,HEK293 cells, Chinese hamster ovary (CHO) cells, and mesenchymal stemcells (MSCs)) to introduce constructs that encode the immunomodulatorycomponent such as a nucleic acid (encoding an ASO) or a protein.Association with cell membrane encompasses binding to the inner or outerlipid leaflet of the membrane and transmembrane insertion into the lipidbilayer. In some instances membrane association is achieved using aprotein (e.g., a scaffold protein or fragment thereof) such as, e.g.,prostaglandin F2 receptor negative regulator (PTGFRN); basigin (BSG);immunoglobulin superfamily member 2 (IGSF2); immunoglobulin superfamilymember 3 (IGSF3); immunoglobulin superfamily member 8 (IGSF8); integrinbeta-1 (ITGB1); integrin alpha-4 (ITGA4); 4F2 cell-surface antigen heavychain (SLC3A2); and a class of ATP transporter proteins (ATP1A1, ATP1A2,ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 that have beenidentified to be highly enriched on the surface of exosomes (asdescribed in co-owned U.S. Pat. No. 10,195,290) which can provide anelement of a fusion protein comprising the scaffold and theimmunomodulatory component. Such extracellular vesicles and exemplarytechniques are described in detail in co-owned U.S. Pat. No. 10,195,290,incorporated herein by reference for all purposes. As described inco-owned U.S. Pat. No. 10,195,290, surface-engineered exosomes can begenerated by chemical and/or physical methods, such as PEG-inducedfusion and/or ultrasonic fusion to introduce these scaffold proteins(and fragments thereof) into the exosomes or producer cells. A complexcan be generated between an exogenous therapeutic protein and thescaffold protein. Alternatively, a fusion protein can be produced byconjugating a scaffold protein and an exogenous therapeutic protein,such as, e.g., an immunomodulating protein, and producing an engineeredexosome containing the complex or fusion protein on the surface, usingthe aforementioned chemical and/or physical methods. A nativefull-length or a biologically active fragment of the therapeutic proteincan be transported to the surface of exosomes by being conjugated to thescaffold protein. Such exosomes can also be obtained from a producercell that comprises the exogenous sequence inserted into a genome of thecell. For example, the exogenous sequence can be inserted into a genomicsite located 3′ or 5′ end of a genomic sequence encoding PTGFRN, BSG,IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2 or ATP transporter. Forexample, the exogenous sequence can be inserted into a genomic sequenceencoding PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2 or ATPtransporter. Association with the cell membrane encompasses binding ofthe immunomodulating component to the internal or external membranesurface, anchoring of the immunomodulating component within the lipidbilayer, or extension of the immunomodulating component through thelipid bilayer. The immunomodulating component can be tethered to ascaffold protein, or expressed as a fusion protein with a scaffoldprotein as described in co-owned U.S. Pat. No. 10,195,290.

The Extracellular Vesicle

In various embodiments, the composition comprises an extracellularvesicle. In certain embodiments, the extracellular vesicle is acell-derived vesicle comprising a membrane that encloses an internalspace, as described in co-owned U.S. Pat. No. 10,195,290.

In various embodiments, the extracellular vesicle can be amembrane-bound vesicle that has a smaller diameter than the cell fromwhich it is derived. In some embodiments, the extracellular vesicle hasa longest dimension between about 20-1000 nm, such as between about20-100 nm, 20-200 nm, 20-300 nm, 20-400 nm, 20-500 nm, 20-600 nm, 20-700nm, 20-800 nm, 20-900 nm, 30-100 nm, 30-200 nm, 30-300 nm, 30-400 nm,30-500 nm, 30-600 nm, 30-700 nm, 30-800 nm, 30-900 nm, 40-100 nm, 40-200nm, 40-300 nm, 40-400 nm, 40-500 nm, 40-600 nm, 40-700 nm, 40-800 nm,40-900 nm, 50-150 nm, 50-500 nm, 50-750 nm, 100-200 nm, 100-500 nm, or500-1000 nm.

In certain embodiments, the extracellular vesicle is an exosome. Incertain embodiments, the extracellular vesicle is a nanovesicle. Incertain embodiments, the extracellular vesicle is an apoptotic body. Incertain embodiments, the extracellular vesicle is a fragment of cell. Incertain embodiments, the extracellular vesicle is a vesicle derived fromcell by direct or indirect manipulation. In certain embodiments, theextracellular vesicle is a vesiculated organelle. In variousembodiments, the extracellular vesicle is a vesicle produced by livingcells.

In some embodiments, the extracellular vesicle is derived from a livingorganism. In some embodiments, the extracellular vesicle is derived froma dead organism. In some embodiments, the extracellular vesicle isderived from an explanted tissue. In some embodiments, the extracellularvesicle is derived from an explanted organ. In some embodiments, theextracellular vesicle is derived from cultured cells. In some of theseembodiments, when the extracellular vesicle is generated in a cellculture system, the extracellular vesicle is further isolated (e.g., byseparating the extracellular vesicle from the cultured cells).Separation can be achieved by sedimentation. For example, theextracellular vesicle can have a specific density between 0.5-2.0,0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.4-1.5,1.0-1.5, 1.5-2.0, and 1.0-2.0 kg/m³. Separation can also be achieved byaffinity purification. For example, the extracellular vesicle can bepurified by binding a population comprising extracellular vesicles to aresin, said resin comprising a plurality of ligands that have specificaffinity for one or more proteins on the surface of the extracellularvesicle. The proteins may be a tetraspanin (e.g., CD63, CD81, CD9), anEWI protein/immunoglobulin superfamily member (e.g., PTGFRN, IGSF8,IGSF3), an integrin (e.g., ITGB1, ITGA4), an ATP transporter protein(e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3,ATP2B4), SLC3A2, BSG, or CD98hc.

In various embodiments, the extracellular vesicle comprises lipids orfatty acids and polypeptides. In certain embodiments, the extracellularvesicle further comprises a sugar. In certain embodiments, theextracellular vesicle further comprises a polynucleotide.

In various embodiments, the extracellular vesicle membrane comprises aninterior surface and an exterior surface and encloses an internal space.In some embodiments, the extracellular vesicle further comprises apayload, such as an immunomodulatory component as described herein. Incertain embodiments, the payload is enclosed within the internal space.In certain embodiments, the payload is displayed on the external surfaceof the extracellular vesicle. In certain embodiments, the payload isspanning the membrane of the extracellular vesicle. In variousembodiments, the payload comprises nucleic acids, proteins,carbohydrates, lipids, small molecules, and/or combinations thereof.Methods for producing extracellular vesicles with payloads includeelectroporation, lyophilization, and genetic engineering of producercells from which extracellular vesicles can be isolated. See, e.g.,co-owned U.S. Pat. No. 10,195,290 and supra. In some embodiments, theextracellular vesicle further comprises a receiver, i.e., a targetingmoiety that can be specific to an organ, a tissue, or a cell asdescribed in co-owned U.S. Pat. No. 10,195,290. Fusion proteins having atargeting moiety are used. For example, fusion proteins can comprisePTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter,or a fragment or a variant thereof, and a targeting moiety. Thetargeting moiety can be used for targeting the exosome to a specificorgan, tissue, or cell for a treatment using the exosome. In someembodiments, the targeting moiety is an antibody or antigen-bindingfragment thereof. Antibodies and antigen-binding fragments thereofinclude whole antibodies, polyclonal, monoclonal and recombinantantibodies, fragments thereof, and further includes single-chainantibodies, humanized antibodies, murine antibodies, chimeric,mouse-human, mouse-primate, primate-human monoclonal antibodies,anti-idiotype antibodies, antibody fragments, such as, e.g., scFv,(scFv) 2, Fab, Fab′, and F(ab′) 2, F(ab1) 2, Fv, dAb, and Fd fragments,diabodies, and antibody-related polypeptides. Antibodies andantigen-binding fragments thereof also includes bispecific antibodiesand multispecific antibodies so long as they exhibit the desiredbiological activity or function.

The Exosome

In various embodiments, the extracellular vesicle is an exosome. Incertain embodiments, the exosome is a small membrane-bound vesiclesecreted by producer cells.

In some embodiments, the exosome from the producer cell has a longestdimension between about 20-300 nm, such as between about 20-290 nm,20-280 nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20-160 nm,20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100 nm, 20-90nm, 20-80 nm, 20-70 nm, 20-60 nm, 20-50 nm, 20-40 nm, 20-30 nm, 30-300nm, 30-290 nm, 30-280 nm, 30-270 nm, 30-260 nm, 30-250 nm, 30-240 nm,30-230 nm, 30-220 nm, 30-210 nm, 30-200 nm, 30-190 nm, 30-180 nm, 30-170nm, 30-160 nm, 30-150 nm, 30-140 nm, 30-130 nm, 30-120 nm, 30-110 nm,30-100 nm, 30-90 nm, 30-80 nm, 30-70 nm, 30-60 nm, 30-50 nm, 30-40 nm,40-300 nm, 40-290 nm, 40-280 nm, 40-270 nm, 40-260 nm, 40-250 nm, 40-240nm, 40-230 nm, 40-220 nm, 40-210 nm, 40-200 nm, 40-190 nm, 40-180 nm,40-170 nm, 40-160 nm, 40-150 nm, 40-140 nm, 40-130 nm, 40-120 nm, 40-110nm, 40-100 nm, 40-90 nm, 40-80 nm, 40-70 nm, 40-60 nm, 40-50 nm, 50-300nm, 50-290 nm, 50-280 nm, 50-270 nm, 50-260 nm, 50-250 nm, 50-240 nm,50-230 nm, 50-220 nm, 50-210 nm, 50-200 nm, 50-190 nm, 50-180 nm, 50-170nm, 50-160 nm, 50-150 nm, 50-140 nm, 50-130 nm, 50-120 nm, 50-110 nm,50-100 nm, 50-90 nm, 50-80 nm, 50-70 nm, 50-60 nm, 60-300 nm, 60-290 nm,60-280 nm, 60-270 nm, 60-260 nm, 60-250 nm, 60-240 nm, 60-230 nm, 60-220nm, 60-210 nm, 60-200 nm, 60-190 nm, 60-180 nm, 60-170 nm, 60-160 nm,60-150 nm, 60-140 nm, 60-130 nm, 60-120 nm, 60-110 nm, 60-100 nm, 60-90nm, 60-80 nm, 60-70 nm, 70-300 nm, 70-290 nm, 70-280 nm, 70-270 nm,70-260 nm, 70-250 nm, 70-240 nm, 70-230 nm, 70-220 nm, 70-210 nm, 70-200nm, 70-190 nm, 70-180 nm, 70-170 nm, 70-160 nm, 70-150 nm, 70-140 nm,70-130 nm, 70-120 nm, 70-110 nm, 70-100 nm, 70-90 nm, 70-80 nm, 80-300nm, 80-290 nm, 80-280 nm, 80-270 nm, 80-260 nm, 80-250 nm, 80-240 nm,80-230 nm, 80-220 nm, 80-210 nm, 80-200 nm, 80-190 nm, 80-180 nm, 80-170nm, 80-160 nm, 80-150 nm, 80-140 nm, 80-130 nm, 80-120 nm, 80-110 nm,80-100 nm, 80-90 nm, 90-300 nm, 90-290 nm, 90-280 nm, 90-270 nm, 90-260nm, 90-250 nm, 90-240 nm, 90-230 nm, 90-220 nm, 90-210 nm, 90-200 nm,90-190 nm, 90-180 nm, 90-170 nm, 90-160 nm, 90-150 nm, 90-140 nm, 90-130nm, 90-120 nm, 90-110 nm, 90-100 nm, 100-300 nm, 110-290 nm, 120-280 nm,130-270 nm, 140-260 nm, 150-250 nm, 160-240 nm, 170-230 nm, 180-220 nm,or 190-210 nm.

In particularly preferred embodiments, the exosome from the producercell described herein has a longest dimension between about 30-100 nm.In another preferred embodiment, the exosome from the producer cell hasa longest dimension between about 20-300 nm. In another preferredembodiment, the exosome from the producer cell has a longest dimensionbetween about 40-200 nm. In another embodiment, a population of theexosomes described herein comprise a population wherein 90% of theexosomes have a longest dimension 20-300 nm. In another embodiment, apopulation of the exosomes described herein comprise a populationwherein 95% of the exosomes have a longest dimension 20-300 nm. Inanother embodiment, a population of the exosomes described hereincomprise a population wherein 99% of the exosomes have a longestdimension 20-300 nm. In another embodiment, a population of the exosomesdescribed herein comprise a population wherein 90% of the exosomes havea longest dimension 40-200 nm. In another embodiment, a population ofthe exosomes described herein comprise a population wherein 95% of theexosomes have a longest dimension 40-200 nm. In another embodiment, apopulation of the exosomes described herein comprise a populationwherein 99% of the exosomes have a longest dimension 40-200 nm. In otherpreferred embodiments, the size of the exosome or population of exosomesdescribed herein is measured according to methods described, infra.

In some embodiments, the exosome is generated by a producer cell. Insome embodiments, the membrane of the exosome comprises one or moremolecules derived from the producer cell. In some embodiments, theexosome is generated in a cell culture system and isolated (e.g., byseparating the exosome from the producer cell). Separation can beachieved by sedimentation. For example, the exosome can have a specificdensity between 0.5-2.0, 0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1,1.1-1.2, 1.2-1.3, 1.4-1.5, 1.0-1.5, 1.5-2.0, and 1.0-2.0 kg/m³.Separation can also be achieved by affinity purification. For example,the extracellular vesicle can be purified by binding a populationcomprising extracellular vesicles to a resin, said resin comprising aplurality of ligands that have specific affinity for one or moreproteins on the surface of the extracellular vesicle. The one or moreproteins on the surface of the extracellular vesicle may be atetraspanin (e.g., CD63, CD81 and/or CD9), an EWI protein/immunoglobulinsuperfamily member (e.g., PTGFRN, IGSF8 and/or IGSF3), an integrin(e.g., ITGB1 and/or ITGA4), an ATP transporter protein (e.g., ATP1A1,ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3 and/or ATP2B4),SLC3A2, BSG, or CD98hc. The protein may additionally have activity as animmunomodulating component displayed on the surface of the exosomes,optionally via fusion to a polypeptide moiety that has a desiredpharmacologic activity, such as, e.g., an antibody, antibody fragment,scFv, etc. with checkpoint inhibition activity. Such antibodies areknown in the art, e.g., Ipilimumab, targeting CTLA-4, NivolumabCemiplimab and Pembrolizumab targeting PD-1, and Atezolizumab, Avelumab,Durvalumab, each targeting PD-L1 (see, e.g., Pardoll D M (March 2012).“The blockade of immune checkpoints in cancer immunotherapy”. NatureReviews. Cancer. 12 (4): 252-64, incorporated herein by reference.

In some embodiments, the exosome membrane comprises an interior surfaceand an exterior surface. In certain embodiments, the interior surfacefaces the inner core of the exosome. In certain embodiments, theexterior surface can be in contact with the endosome, the multivesicularbodies, or the membrane/cytoplasm of a producer cell or a target cell.

In some embodiments, the exosome membrane comprises lipids and fattyacids. In some embodiments, the exosome membrane comprisesphospholipids, glycolipids, fatty acids, sphingolipids,phosphoglycerides, sterols, cholesterols, and phosphatidylserines. Insome embodiments, the lipid and fatty acid can be one or more of thoselisted in Table 1.

In certain embodiments, the exosome comprises a lipid bilayer composedof an inner leaflet and an outer leaflet. The composition of the innerand outer leaflet can be determined by transbilayer distribution assaysknown in the art, see e.g., Kuypers et al. Biohim Biophys Acta 1985819:170. In some embodiments, the composition of the outer leaflet isbetween approximately 70-90% choline phospholipids, betweenapproximately 0-15% acidic phospholipids, and between approximately5-30% phosphatidylethanolamine. In some embodiments, the composition ofthe inner leaflet is between approximately 15-40% choline phospholipids,between approximately 10-50% acidic phospholipids, and betweenapproximately 30-60% phosphatidylethanolamine.

In some embodiments, the exosome membrane further comprises one or morepolypeptides. In certain embodiments, the exosome comprises one or morepolypeptide selected from the following list, including but not limitedto, spectrin, myosin-like polypeptide, band 3, SLC4A1, actin, actin-likepolypeptide, glyceraldehyde 3-P dehydrogenase (G3PD), tetraspanins(e.g., CD63, CD81 and/or CD9), Alix and TSG101, integrins (e.g., ITGB1and/or ITGA4), selectins, CR1, TNFRI, proteolytic enzymes,glycosylphosphatidylinositol (GPI)-linked proteins or histones, EWIprotein/immunoglobulin superfamily members (e.g., PTGFRN, IGSF8 and/orIGSF3), ATP transporter proteins (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4,ATP1B3, ATP2B1, ATP2B2, ATP2B3 and/or ATP2B4), SLC3A2, BSG, or CD98hc.)In some embodiments, the exosome comprises at least one polypeptideselected from Table 2.

In some embodiments, the exosome comprises polypeptides on its surface.In some embodiments, the exosome is modified to contain the one or morepolypeptides. In some embodiments, the producer cell is modified tocontain the one or more polypeptides. In some embodiments, the producercell naturally contains the one or more polypeptides and exosomesderived therefrom also contain the polypeptides. The levels of anydesired surface marker can be modified directly on the exosome (e.g., bycontacting the complex with recombinantly produced polypeptides to bringabout insertion in or conjugation to the membrane of the complex).Alternatively or in addition, the levels of any desired surface markercan be modified directly on the producer cell (e.g., by contacting thecomplex with recombinantly produced polypeptides to bring aboutinsertion in or conjugation to the membrane of the cell). Alternatively,the producer cell can be modified by transducing an exogenous nucleicacid into the producer cell to express a desired surface marker. Thesurface marker can already be naturally present on the producer cell, inwhich case the exogenous construct can lead to overexpression of themarker and increased concentration of the marker in or on the producercell. Alternatively, a naturally expressed surface marker can be removedfrom the producer cell (e.g., by inducing gene silencing in the producercell). The polypeptides can confer different functionalities to theexosome (e.g., specific targeting capabilities, delivery functions(e.g., fusion molecules), enzymatic functions, increased or decreasedhalf-life in vivo, etc.). In some embodiments, the polypeptides include,but are not limited to CD47, CD55, CD49, CD40, CD133, CD59, glypican-1,CD9, CD63, CD81, integrins, selectins, lectins, and cadherins.

In specific embodiments, the exosomes comprise one or more polypeptideson their surface, wherein said polypeptides are selected from a group ofproteins that was recently identified to be enriched on the surface ofexosomes (described in detail in co-owned U.S. Patent Application62/550,543, and PCT/US2018/048026, which is incorporated herein byreference in their entireties). This group of polypeptides includesprostaglandin F2 receptor negative regulator (PTGFRN); basigin (BSG);immunoglobulin superfamily member 3 (IGSF3); immunoglobulin superfamilymember 8 (IGSF8); integrin beta-1 (ITGB1); integrin alpha-4 (ITGA4); 4F2cell-surface antigen heavy chain (SLC3A2); and a class of ATPtransporter proteins (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1,ATP2B2, ATP2B3, ATP2B4)).

In various embodiments, the one or more polypeptides on the exosomesurface comprises an antibody or an antigen-binding fragment. Theantibody or antigen-binding fragment can be derived from naturalsources, or partly or wholly synthetically produced. In someembodiments, the antibody is a monoclonal antibody. In some of theseembodiments, the monoclonal antibody is an IgG antibody. In certainembodiments, the monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4. Insome other embodiments, the antibody is a polyclonal antibody. Incertain embodiments, the antigen-binding fragment is selected from Fab,Fab′, and F(ab′)₂, F(ab1)₂, Fv, dAb, and Fd fragments. In certainembodiments, the antigen-binding fragment is an scFv or (scFv)₂fragment. In certain other embodiments, the antibody or antigen-bindingfragment is a Nanobody® (single-domain antibody). In some embodiments,the antibody or antigen-binding fragment is a bispecific ormultispecific antibody. In various embodiments, the antibody orantigen-binding fragment thereof binds to mesothelin.

In some embodiments, the exosomes comprise on their surface a fusionprotein comprising (1) PTGFRN or a fragment thereof and (2) an antibodyor antigen-binding fragment thereof, wherein the antibody or antigenbinding fragment thereof binds to PD-1, PD-1L, CSF1-R, or otherimmunomodulatory component.

In some embodiments, the exosome membrane further comprises one or morepolysaccharide, such as glycan.

In some embodiments, the exosome delivers the payload (therapeuticagent) to a target. The payload is a therapeutic agent that acts on atarget (e.g., a target cell) that is contacted with the exosome. Incertain embodiments, the payload is an immunomodulating component, e.g.,an inhibitory RNA. Contacting can occur in vitro or in a subject.Payloads that can be introduced into an exosome and/or a producer cellinclude therapeutic agents such as, nucleotides (e.g., nucleotidescomprising a detectable moiety or a toxin or that disrupttranscription), nucleic acids (e.g., DNA or mRNA molecules that encode apolypeptide such as an enzyme, or RNA molecules that have regulatoryfunction such as miRNA, dsDNA, lncRNA, or siRNA), amino acids (e.g.,amino acids comprising a detectable moiety or a toxin that disrupttranslation), polypeptides (e.g., enzymes), lipids, carbohydrates, smallmolecules (e.g., small molecule drugs and toxins), and combinationsthereof. In certain embodiments, the exosome delivers more than onetherapeutic agent. In certain embodiments, the therapeutic agents areone or more nucleic acids that inhibits one or more target genes. Incertain embodiments, the therapeutic agents comprise an antibody and anucleic acid. In certain embodiments, the therapeutic agents comprise anucleic acid and a small molecule. In certain embodiments, thetherapeutic agent comprises a CSF1R inhibitor, such as clinicalcandidates Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945,Emactuzumab, AMG820, and IMC-CS4 and others described in e.g., CannarileM A, Weisser M, Jacob W, Jegg A M, Ries C H, Rüttinger D (2017).“Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancertherapy”. Journal for Immunotherapy of Cancer. 5 (1): 53; see also,e.g., Patel S, Player M R (2009). “Colony-stimulating factor-1 receptorinhibitors for the treatment of cancer and inflammatory disease”. CurrTop Med Chem. 9: 599-610, Cabiralizumab (cabira; FPA-008) which is amonoclonal antibody and is in early clinical trials for metastaticpancreatic cancer (see A phase I/II dose escalation and expansion studyof cabiralizumab (cabira; FPA-008), an anti-CSF1R antibody, intenosynovial giant cell tumor (TGCT, diffuse pigmented villonodularsynovitis D-PVNS; A Study of Cabiralzumab Given by Itself or WithNivolumab in Advanced Cancer or Cancer That Has Spread; and NovelCombination Shows Promising Responses in Pancreatic Cancer November2017) In certain embodiments, the therapeutic agent comprises an immunecheckpoint inhibitor. In certain embodiments, the immune checkpointinhibitor in an antibody such as, e.g., Ipilimumab, targeting CTLA-4,Nivolumab Cemiplimab and Pembrolizumab targeting PD-1, and Atezolizumab,Avelumab, Durvalumab, each targeting PD-L1

The exosome can interact with the target cell via membrane fusion anddeliver payloads (e.g., therapeutic agents) in an exosome composition tothe surface or cytoplasm of a target cell. In some embodiments, membranefusion occurs between the exosome and the plasma membrane of a targetcell. In other embodiments, membrane fusion occurs between the exosomeand an endosomal membrane of a target cell.

In some embodiments, the exosome comprises a receiver polypeptide. Thereceiver polypeptide can be synthetic. In some embodiments, the receiverpolypeptide is introduced into the producer cell (e.g., an exogenousnucleic acid that encodes the receiver polypeptide is introduced intothe producer cell) or a recombinant receiver polypeptide that is madeoutside the producer cell (e.g., synthesized by a protein expressionsystem). In some embodiments, the receiver polypeptide (e.g., arecombinantly produced polypeptide) is introduced into the exosomedirectly (e.g., after the exosome is isolated from the producer cell).In some embodiments, the receiver polypeptide can be on the surface ofthe exosomes. In some embodiments, the receiver polypeptide is capableof targeting the exosome to a specific target (e.g., a target such as apathogen, a metabolite, a protein, a polypeptide complex or a cell suchas non-functional cell or cancer cell) that circulates in thecirculatory system of the subject, such as the blood, or a target thatresides in a tissue (such as a diseased tissue).

In some embodiments, the exosome is synthetic. That is to say thatmodifications are made to the exosomes after their recovery from theproducer cell to add additional components. For example, the exosome cancomprise a payload, such as, e.g., a therapeutic polypeptide, nucleicacid (such as DNA or RNA) or other polynucleotide, polysaccharide orglycan, lipid or fatty acid, large biologic, small molecule or toxin notfound in exosomes upon recovery from the producer cell. In someembodiments, the exosome is modified (e.g., by introducing a payload orotherwise modifying the content of the complex, such as by changing theprotein, lipid or glycan content of the membrane). For example, exosomesare first isolated from a producer cell and then modified as desired,thereby generating synthetic exosomes. In some embodiments, the producercell is modified. For example, an exogenous nucleic acid, an exogenouspolypeptide or small molecule or toxin can be introduced into theproducer cell. Alternatively or in addition, the producer cell canotherwise be modified (e.g., by modifying the cellular or membranecontent, such as by changing the lipid or glycan content of the cellmembrane). Exosomes generated from the modified producer cells compriseone or more of the modifications of the producer cell. The processproduces synthetic exosomes. In some embodiments, both the producer celland the exosome isolated from the producer cell are modified asdescribed herein.

Nanovesicle

In various embodiments, the extracellular vesicle is a nanovesicle. Incertain embodiments, the nanovesicle is a cell-derived small vesiclecomprising a membrane that encloses an internal space, and which isgenerated from the cell by direct or indirect manipulation such that thenanovesicle would not be produced by the cell without the manipulation.Appropriate manipulations of the cell include but are not limited toserial extrusion, treatment with alkaline solutions, sonication, orcombinations thereof and can, in some instances, result in thedestruction of the producer cell.

In various embodiments, the nanovesicle has a longest dimension betweenabout 20-250 nm, such as between about 20-100 nm, 20-150 nm, 20-200 nm,30-100 nm, 30-150 nm, 30-200 nm, 30-250 nm, 40-100 nm, 40-150 nm, 40-200nm, 40-250 nm, 50-100 nm, 50-150 nm, 50-200 nm, 50-250 nm, 100-200 nm,or 150-250 nm.

In various embodiments, the nanovesicle is derived from a producer cell.In certain embodiments, the nanovesicle is generated from a producercell by direct or indirect manipulation. Appropriate manipulationsinclude but are not limited to serial extrusion, treatment with alkalinesolutions, sonication, or combinations thereof. In some of theseembodiments, the manipulation can result in the destruction of theproducer cell. In some preferred embodiments, the population of thenanovesicle is substantially free of vesicles that are derived fromproducer cells by way of direct budding from the plasma membrane orfusion of the late endosome with the plasma membrane.

In some embodiments, the nanovesicle is isolated from the producer cellbased on its size, density, biochemical parameters, or a combinationthereof. In certain embodiments, the isolation can be achieved bysedimentation. For example, the nanovesicle can have a specific densitybetween 0.5-2.0, 0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1, 1.1-1.2,1.2-1.3, 1.4-1.5, 1.0-1.5, 1.5-2.0, and 1.0-2.0 kg/m³.

In various embodiments, the nanovesicle comprises lipids or fatty acidsand polypeptides. In certain embodiments, the nanovesicle furthercomprises a sugar. In certain embodiments, the nanovesicle furthercomprises a polynucleotide. In some embodiments, the nanovesicle furthercomprises a receiver. In some embodiments, the nanovesicle furthercomprises a payload. In some of these embodiments, the payload comprisesnucleic acids, proteins, carbohydrates, lipids, small molecules, and/orcombinations thereof.

The Immunomodulating Component

In an aspect, the extracellular vesicle, e.g., an exosome, comprises atleast one immunomodulating component that increases macrophagepolarization from an M2 to an M1 phenotype. In an aspect the increasefrom an M2 to an M1 phenotype is assessed with respect to a referencesample comprising a population of M2 macrophages. In an aspect theincrease achieved by the extracellular vesicles of the presentdisclosure is greater than or equal to that achieved by an equimolaramount of the immunomodulating component alone. In certain embodiments,the immunomodulating component is a polynucleotide that increasesmacrophage polarization. In certain embodiments, the polynucleotide is anucleic acid that inhibits at least one gene, such as, e.g.,protooncogenes and other type of genes, the inhibition of which promoteincreased polarization from an M2 to an M1 phenotype, including by wayof example, but not limitation, the following genes: KRAS, HRAS, NRAS,HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC,c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, Glutaminase, CEBP/β, Pi3Kγ, andPKM2. In certain embodiments, combinations of polynucleotide andproteinaceous immunomodulating components are contemplated, includingproteinaceous immunomodulating components such as, e.g., antibodies orantibody fragments that target PD-1, PD-L1, CTLA-4 (such as, e.g.,Ipilimumab, targeting CTLA-4, Nivolumab Cemiplimab and Pembrolizumabtargeting PD-1, and Atezolizumab, Avelumab, Durvalumab, each targetingPD-L1), CSF1R inhibitors (such as, e.g., such as clinical candidatesPexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab,AMG820, and IMC-CS4 and others described in e.g., Cannarile M A, WeisserM, Jacob W, Jegg A M, Ries C H, Rittinger D (2017). “Colony-stimulatingfactor 1 receptor (CSF1R) inhibitors in cancer therapy”. Journal forImmunotherapy of Cancer. 5 (1): 53; see also, e.g., Patel S, Player M R(2009). “Colony-stimulating factor-1 receptor inhibitors for thetreatment of cancer and inflammatory disease”. Curr Top Med Chem. 9:599-610, Cabiralizumab (cabira; FPA-008) which is a monoclonal antibodyand is in early clinical trials for metastatic pancreatic cancer (see Aphase I/II dose escalation and expansion study of cabiralizumab (cabira;FPA-008), an anti-CSF1R antibody, in tenosynovial giant cell tumor(TGCT, diffuse pigmented villonodular synovitis D-PVNS; A Study ofCabiralzumab Given by Itself or With Nivolumab in Advanced Cancer orCancer That Has Spread; and Novel Combination Shows Promising Responsesin Pancreatic Cancer November 2017, and other immunomodulatorycomponents useful in the treatment of cancer and inflammatoryconditions.

In some of these embodiments, the nucleic acid includes, but is notlimited to, an mRNA, a miRNA, an siRNA, an antisense RNA, an shRNA, alncRNA, and a dsDNA. In some embodiments, the nucleic acid is an RNA(e.g., an mRNA, a miRNA, an siRNA, an antisense RNA, an shRNA, or anlncRNA). In some of these embodiments, when the polynucleotide is anmRNA, it can be translated into a desired polypeptide. In someembodiments, the polynucleotide is a microRNA (miRNA), pri-miRNA, orpre-miRNA molecule. In some of these embodiments, the miRNA is deliveredto the cytoplasm of the target cell, such that the miRNA molecule cansilence a native mRNA in the target cell. In some embodiments, thepolynucleotide is a small interfering RNA (siRNA) or a short hairpin RNA(shRNA) capable of interfering with the expression of an oncogene orother dysregulating polypeptides. In some of these embodiments, thesiRNA is delivered to the cytoplasm of the target cell, such that thesiRNA molecule can silence a native mRNA in the target cell. In someembodiments, the polynucleotide is an antisense RNA that iscomplementary to an mRNA. In some embodiments, the polynucleotide is along non-coding RNA (lncRNA) capable of regulating gene expression andmodulating diseases. In some embodiments, the polynucleotide is anantisense oligonucleotide (ASO). In various embodiments, the ASO is asingle-stranded or double-stranded DNA, RNA, or DNA/RNA hybrid. See,e.g., Antisense Oligodeoxynucleotides and Antisense RNA: NovelPharmacological and Therapeutic Agents, CRC Press, Boca Raton, Fla.,1997, incorporated herein by reference for all purposes.

In some embodiments, the polynucleotide is a DNA that can be transcribedinto an RNA. In some of these embodiments, the transcribed RNA can betranslated into a desired polypeptide. In certain embodiments, thenucleic acid inhibits at least one gene consisting of: KRAS, HRAS, NRAS,HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC,c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, CEBP/β, Pi3Kγ, Glutaminase, andPKM2. In certain embodiments, the nucleic acid inhibits at least onegene consisting of: STAT3, STAT6, CEBP/β, Pi3Kγ, KRAS, and HIF1-alpha.In certain embodiments, the nucleic acid is an inhibitory RNA thatinhibits at least one gene consisting of: KRAS, HRAS, NRAS, HIF1-alpha,HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1,A2AB, IDO, TDO, Arginase, CEBP/β, Pi3Kγ, Glutaminase, and PKM2. Incertain embodiments, the nucleic acid is an inhibitory RNA that inhibitsat least one gene consisting of: STAT3, STAT6, CEBP/β, Pi3Kγ, KRAS, andHIF1-alpha. In certain embodiments, the nucleic acid is an antisenseoligonucleotide (ASO) that inhibits at least one gene consisting of:KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3,STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, CEBP/β, Pi3Kγ,Glutaminase, and PKM2. In certain embodiments, the nucleic acid is anantisense oligonucleotide (ASO) that inhibits at least one geneconsisting of: STAT3, STAT6, CEBP/β, Pi3Kγ, KRAS, and HIF1-alpha. Incertain embodiments, the nucleic acid inhibits the human KRASproto-oncogene. In certain embodiments, the nucleic acid is aninhibitory RNA that inhibits the human KRAS proto-oncogene. In certainembodiments, the nucleic acid inhibits STAT3.

In some embodiments of the present invention, the nucleic acid is aknown antisense oligonucleotide (ASO), examples of which are listed inTable 0. In certain embodiments the ASO comprises a sequence at least90% to 99% identical to a known ASO, e.g., those listed in Table 0. Incertain embodiments the ASO inhibits STAT3. In various embodiments, theASO comprises a sequence at least 90% to 99% identical toTAAGCTGATAATTCAACTCA (SEQ ID NO:1). In certain embodiments, the ASOcomprises a sequence at least 90% identical to SEQ ID NO:1. In certainembodiments, the ASO comprises a sequence at least 91% identical to SEQID NO:1. In certain embodiments, the ASO comprises a sequence at least92% identical to SEQ ID NO:1. In certain embodiments, the ASO comprisesa sequence at least 93% identical to SEQ ID NO:1. In certainembodiments, the ASO comprises a sequence at least 94% identical to SEQID NO:1. In certain embodiments, the ASO comprises a sequence at least95% identical to SEQ ID NO:1. In certain embodiments, the ASO comprisesa sequence at least 96% identical to SEQ ID NO:1. In certainembodiments, the ASO comprises a sequence at least 97% identical to SEQID NO:1. In certain embodiments, the ASO comprises a sequence at least98% identical to SEQ ID NO:1. In certain embodiments, the ASO comprisesa sequence at least 99% identical to SEQ ID NO:1. In certainembodiments, the ASO comprises a sequence of SEQ ID NO:1. In someembodiments, the ASO is modified by MOE, O-methyl, or LNA chemistry. Insome embodiments, the ASO further comprises a cholesterol tag at the 5′or 3′ end.

In some embodiment, the nucleic acid is an antisense oligonucleotide(ASO) that inhibits STAT6. In various embodiments, the ASO comprises asequence at least 90% to 99% identical to TGAGCGAATGGACAGGTCTT (SEQ IDNO:2). In certain embodiments, the ASO comprises a sequence at least 90%identical to SEQ ID NO:2. In certain embodiments, the ASO comprises asequence at least 91% identical to SEQ ID NO:2. In certain embodiments,the ASO comprises a sequence at least 92% identical to SEQ ID NO:2. Incertain embodiments, the ASO comprises a sequence at least 93% identicalto SEQ ID NO:2. In certain embodiments, the ASO comprises a sequence atleast 94% identical to SEQ ID NO:2. In certain embodiments, the ASOcomprises a sequence at least 95% identical to SEQ ID NO:2. In certainembodiments, the ASO comprises a sequence at least 96% identical to SEQID NO:2. In certain embodiments, the ASO comprises a sequence at least97% identical to SEQ ID NO:2. In certain embodiments, the ASO comprisesa sequence at least 98% identical to SEQ ID NO:2. In certainembodiments, the ASO comprises a sequence at least 99% identical to SEQID NO:2. In certain embodiments, the ASO comprises a sequence of SEQ IDNO:2. In some embodiments, the ASO is modified by MOE, O-methyl, or LNAchemistry. In some embodiments, the ASO further comprises a cholesteroltag at the 5′ or 3′ end.

In some embodiment, the nucleic acid is an antisense oligonucleotide(ASO) that inhibits CebpB. In various embodiments, the ASO comprises asequence at least 90% to 99% identical to TGGATTTAAAGGCAGGCGGC (SEQ IDNO:3). In certain embodiments, the ASO comprises a sequence at least 90%identical to SEQ ID NO:3. In certain embodiments, the ASO comprises asequence at least 91% identical to SEQ ID NO:3. In certain embodiments,the ASO comprises a sequence at least 92% identical to SEQ ID NO:3. Incertain embodiments, the ASO comprises a sequence at least 93% identicalto SEQ ID NO:3. In certain embodiments, the ASO comprises a sequence atleast 94% identical to SEQ ID NO:3. In certain embodiments, the ASOcomprises a sequence at least 95% identical to SEQ ID NO:3. In certainembodiments, the ASO comprises a sequence at least 96% identical to SEQID NO:3. In certain embodiments, the ASO comprises a sequence at least97% identical to SEQ ID NO:3. In certain embodiments, the ASO comprisesa sequence at least 98% identical to SEQ ID NO:3. In certainembodiments, the ASO comprises a sequence at least 99% identical to SEQID NO:3. In certain embodiments, the ASO comprises a sequence of SEQ IDNO:3. In some embodiments, the ASO is modified by MOE, O-methyl, or LNAchemistry. In some embodiments, the ASO further comprises a cholesteroltag at the 5′ or 3′ end.

In some embodiment, the nucleic acid is an antisense oligonucleotide(ASO) that inhibits Pi3Kγ. In various embodiments, the ASO comprises asequence at least 90% to 99% identical to TTGGGTAAAGTCGTGCAGCA (SEQ IDNO:4). In certain embodiments, the ASO comprises a sequence at least 90%identical to SEQ ID NO:4. In certain embodiments, the ASO comprises asequence at least 91% identical to SEQ ID NO:4. In certain embodiments,the ASO comprises a sequence at least 92% identical to SEQ ID NO:4. Incertain embodiments, the ASO comprises a sequence at least 93% identicalto SEQ ID NO:4. In certain embodiments, the ASO comprises a sequence atleast 94% identical to SEQ ID NO:4. In certain embodiments, the ASOcomprises a sequence at least 95% identical to SEQ ID NO:4. In certainembodiments, the ASO comprises a sequence at least 96% identical to SEQID NO:4. In certain embodiments, the ASO comprises a sequence at least97% identical to SEQ ID NO:4. In certain embodiments, the ASO comprisesa sequence at least 98% identical to SEQ ID NO:4. In certainembodiments, the ASO comprises a sequence at least 99% identical to SEQID NO:4. In certain embodiments, the ASO comprises a sequence of SEQ IDNO:4. In some embodiments, the ASO is modified by MOE, O-methyl, or LNAchemistry. In some embodiments, the ASO further comprises a cholesteroltag at the 5′ or 3′ end.

In some embodiment, the nucleic acid is an antisense oligonucleotide(ASO) that inhibits HIF1α. In various embodiments, the ASO comprises asequence at least 90% to 99% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 90% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence at least91% identical to SEQ ID NO:5. In certain embodiments, the ASO comprisesa sequence at least 92% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 93% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence at least94% identical to SEQ ID NO:5. In certain embodiments, the ASO comprisesa sequence at least 95% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 96% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence at least97% identical to SEQ ID NO:5. In certain embodiments, the ASO comprisesa sequence at least 98% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 99% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence of SEQ IDNO:5. In some embodiments, the ASO is modified by MOE, O-methyl, or LNAchemistry. In some embodiments, the ASO further comprises a cholesteroltag at the 5′ or 3′ end. In some embodiment, the nucleic acid is anantisense oligonucleotide (ASO) that inhibits HIF1α. In variousembodiments, the ASO comprises a sequence at least 90% to 99% identicalto GTGCAGTATTGTAGCCAGGC (SEQ ID NO:5). In certain embodiments, the ASOcomprises a sequence at least 90% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 91% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence at least92% identical to SEQ ID NO:5. In certain embodiments, the ASO comprisesa sequence at least 93% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 94% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence at least95% identical to SEQ ID NO:5. In certain embodiments, the ASO comprisesa sequence at least 96% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence at least 97% identical to SEQID NO:5. In certain embodiments, the ASO comprises a sequence at least98% identical to SEQ ID NO:5. In certain embodiments, the ASO comprisesa sequence at least 99% identical to SEQ ID NO:5. In certainembodiments, the ASO comprises a sequence of SEQ ID NO:5. In someembodiments, the ASO is modified by MOE, O-methyl, or LNA chemistry. Insome embodiments, the ASO further comprises a cholesterol tag at the 5′or 3′ end.

In some embodiment, the nucleic acid is an antisense oligonucleotide(ASO) that inhibits Kras. In various embodiments, the ASO comprises asequence at least 90% to 99% identical to GTAGCATGTAAATATAGCCC (SEQ IDNO:6). In certain embodiments, the ASO comprises a sequence at least 90%identical to SEQ ID NO:6. In certain embodiments, the ASO comprises asequence at least 91% identical to SEQ ID NO:6. In certain embodiments,the ASO comprises a sequence at least 92% identical to SEQ ID NO:6. Incertain embodiments, the ASO comprises a sequence at least 93% identicalto SEQ ID NO:6. In certain embodiments, the ASO comprises a sequence atleast 94% identical to SEQ ID NO:6. In certain embodiments, the ASOcomprises a sequence at least 95% identical to SEQ ID NO:6. In certainembodiments, the ASO comprises a sequence at least 96% identical to SEQID NO:6. In certain embodiments, the ASO comprises a sequence at least97% identical to SEQ ID NO:6. In certain embodiments, the ASO comprisesa sequence at least 98% identical to SEQ ID NO:6. In certainembodiments, the ASO comprises a sequence at least 99% identical to SEQID NO:6. In certain embodiments, the ASO comprises a sequence of SEQ IDNO:6. In some embodiments, the ASO is modified by MOE, O-methyl, or LNAchemistry. In some embodiments, the ASO further comprises a cholesteroltag at the 5′ or 3′ end.

In certain embodiments, the composition comprises an extracellularvesicle, e.g., an exosome, further comprising an additionalimmunomodulating component that is a small molecule, antibody orantibody fragment known to promote macrophage polarization. In certainembodiments, the additional immunomodulating component is a ColonyStimulating Factor 1 Receptor (CSF1R) inhibitor.

In certain embodiments, the composition comprises an additionalimmunomodulating component that has anti-tumor activity. In someembodiments, the additional immunomodulating component regulates theinnate immune response. In some of these embodiments, the additionalimmunomodulating component targets the natural killer cells. In someother embodiments, the additional immunomodulating component regulatesthe adaptive immune response. In some of these embodiments, theadditional immunomodulating component targets the cytotoxic T cells.

In some embodiments, the additional immunomodulating component islocated on the surface of the extracellular vesicle. In someembodiments, the additional immunomodulating component is located insidethe extracellular vesicle. In some embodiments, the additionalimmunomodulating component is located both on the surface of and insidethe extracellular vesicle.

In some embodiments, the additional immunomodulating component isexpressed in the producer cell in its full-length form. In otherembodiments, the additional immunomodulating component is expressed as atranslational fusion protein to an exosome surface protein, whichresults in a higher level of expression of the biologically activeportion of the immunomodulating compound on the surface of the exosome.In some embodiments, the additional immunomodulating compound is asoluble protein that is expressed as a translational fusion protein toan exosome surface protein, such that said soluble protein is retainedon the surface of the exosome.

In some embodiments, the additional immunomodulating component is aninhibitor for a negative checkpoint regulator, such as, e.g., A2AR,B7-H3 (CD276), B7-H4 (VTCN1), BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1,TIM-3, VISTA, SIGLEC7 (CD328) and SIGLEC9 (CD329). In some embodiments,the additional immunomodulating component is an inhibitor for a bindingpartner of a negative checkpoint regulator, such as, e.g., PD-L1 andPD-L2.

In certain embodiments, the additional immunomodulating component is aninhibitor of cytotoxic T-lymphocyte-associate protein 4 (CTLA-4). Insome of these embodiments, the CTLA-4 inhibitor is a monoclonal antibodyof CTLA-4. In certain embodiments, the inhibitor is a fragment of amonoclonal antibody of CTLA-4. In certain embodiments, the antibodyfragment is a scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂, F(ab1)₂, Fv, dAb,or Fd of a monoclonal antibody of CTLA-4. In certain embodiments, theinhibitor is a nanobody, a bispecific antibody, or a multispecificantibody against CTLA-4. In some specific embodiments, the monoclonalantibody is ipilimumab. In some specific embodiments, the monoclonalantibody is tremelimumab.

In certain embodiments, the additional immunomodulating component is aninhibitor of programmed cell death protein 1 (PD-1). In certainembodiments, the additional immunomodulating component is an inhibitorof programmed death-ligand 1 (PD-L1). In certain embodiments, theadditional immunomodulating component is an inhibitor of programmeddeath-ligand 2 (PD-L2). In some embodiments, the inhibitor of PD-1,PD-L1, or PD-L2 is a monoclonal antibody of PD-1, PD-L1, or PD-L2. Incertain embodiments, the inhibitor is a fragment of a monoclonalantibody of PD-1, PD-L1, or PD-L2. In certain embodiments, the antibodyfragment is a scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂, F(ab1)₂, Fv, dAb,or Fd of a monoclonal antibody of PD-1, PD-L1, or PD-L2. In certainembodiments, the inhibitor is a nanobody, a bispecific antibody, or amultispecific antibody against PD-1, PD-L1, or PD-L2. In some specificembodiments, the monoclonal antibody is nivolumab. In some specificembodiments, the monoclonal antibody is pembrolizumab. In some specificembodiments, the monoclonal antibody is pidilizumab. In some specificembodiments, the monoclonal antibody is atezolizumab. In some specificembodiments, the monoclonal antibody is avelumab.

In certain embodiments, the additional immunomodulating component is aninhibitor of lymphocyte-activated gene 3 (LAG3). In some of theseembodiments, the inhibitor of LAG3 is a monoclonal antibody of LAG3,such as, e.g., BMS-986016 (see Clinical trial number NCT01968109 for“Safety Study of Anti-LAG-3 With and Without Anti-PD-1 in the Treatmentof Solid Tumors” at ClinicalTrials.gov).

In certain embodiments, the additional immunomodulating component is aninhibitor of T-cell immunoglobulin mucin-containing protein 3 (TIM-3).In certain embodiments, the additional immunomodulating component is aninhibitor of B and T lymphocyte attenuator (BTLA). In certainembodiments, the additional immunomodulating component is an inhibitorof T cell immunoreceptor with Ig and ITIM domains (TIGIT). In certainembodiments, the additional immunomodulating component is an inhibitorof V-domain Ig suppressor of T cell activation (VISTA). In certainembodiments, the additional immunomodulating component is an inhibitorof adenosine A2a receptor (A2aR). In certain embodiments, the additionalimmunomodulating component is an inhibitor of killer cell immunoglobulinlike receptor (KIR). In certain embodiments, the additionalimmunomodulating component is an inhibitor of indoleamine2,3-dioxygenase (IDO). In certain embodiments, the additionalimmunomodulating component is an inhibitor of CD20, CD39, or CD73.

In some embodiments, the additional immunomodulating component is anactivator for a positive co-stimulatory molecule. In some embodiments,the additional immunomodulating component is an activator for a bindingpartner of a positive co-stimulatory molecule.

In some embodiments, the additional immunomodulating component is anactivator of a TNF receptor superfamily member, such as an agonisticantibody or a natural ligand or a TNF receptor superfamily member. Incertain embodiments, the TNF receptor superfamily member is selectedfrom the group consisting of CD120a, CD20b, CD18, OX40, CD40, Fasreceptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4,RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269,GITR, TROY, CD358, TRAMP, and XEDAR. In some embodiments, the additionalimmunomodulating component is a TNF superfamily member. In certainembodiments, the TNF superfamily member is selected from the groupconsisting of: TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L,Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF,BDNF, NT-3, NT-4, GITR ligand, and EDA-2.

In certain embodiments, the additional immunomodulating component is anactivator of TNF Receptor Superfamily Member 4 (OX40). In some of theseembodiments, the activator of OX40 is an agonist antibody of OX40. Insome other of these embodiments, the activator of OX40 is OX40 ligand(OX40L).

In certain embodiments, the additional immunomodulating component is anactivator of CD27. In some of these embodiments, the activator of CD27is an agonist antibody of CD27. In some other of these embodiments, theactivator of CD27 is CD27 ligand (CD27L).

In certain embodiments, the additional immunomodulating component is anactivator of CD40. In some of these embodiments, the activator of CD40is an agonist antibody of CD40. In some other of these embodiments, theactivator of CD40 is CD40 ligand (CD40L).

In certain embodiments, the additional immunomodulating component is anactivator of glucocorticoid-induced TNFR-related protein (GITR). In someof these embodiments, the activator of GITR is an agonist antibody ofGITR. In some other of these embodiments, the activator of GITR is anatural ligand of GITR.

In certain embodiments, the additional immunomodulating component is anactivator of 4-1BB. In some of these embodiments, the activator of 4-1BBis an agonist antibody of 4-1BB. In some other of these embodiments, theactivator of 4-1BB is a natural ligand of 4-1BB.

In some embodiments, the additional immunomodulating component is Fasreceptor (Fas). In some of these embodiments, the Fas receptor isdisplayed on the surface of the extracellular vesicle. In some otherembodiments, the additional immunomodulating component is Fas ligand(FasL). In some of these embodiments, the Fas ligand is displayed on thesurface of the extracellular vesicle. In certain embodiments, theadditional immunomodulating component is an antibody of Fas receptor. Incertain embodiments, the additional immunomodulating component is anantibody of Fas ligand.

In some embodiments, the additional immunomodulating component is anactivator of a CD28-superfamily co-stimulatory molecule. In certainembodiments, the CD28-superfamily co-stimulatory molecule is ICOS orCD28. In certain embodiments, the additional immunomodulating componentis ICOSL, CD80, or CD86.

In certain embodiments, the additional immunomodulating component is anactivator of inducible T cell co-stimulator (ICOS). In some of theseembodiments, the activator of ICOS is an agonist antibody of ICOS. Insome other of these embodiments, the activator of ICOS is ICOS ligand(ICOSL).

In certain embodiments, the additional immunomodulating component is anactivator of CD28. In some of these embodiments, the activator of CD28is an agonist antibody of CD28. In some other of these embodiments, theactivator of CD28 is a natural ligand of CD28. In certain embodiments,the ligand of CD28 is CD80.

In certain embodiments, the additional immunomodulating component is acytokine. In some embodiments, the cytokine is a soluble cytokine thathas been translationally fused to an exosome surface protein or fragmentthereof. In some embodiments, the cytokine is IL-2. In some embodiments,the cytokine is IL-7. In some embodiments, the cytokine is IL-12. Insome embodiments, the cytokine is IL-15.

In some embodiments, the additional immunomodulating component is aT-cell receptor (TCR) or a derivative thereof. In certain embodiments,the additional immunomodulating component is a TCR α-chain or aderivative thereof. In certain embodiments, the additionalimmunomodulating component is a TCR β-chain or a derivative thereof. Insome embodiments, the additional immunomodulating component is aco-receptor of the T-cell or a derivative thereof.

In some embodiments, the additional immunomodulating component is atumor antigen. In certain embodiments, the tumor antigen is selectedfrom the group consisting of: alpha-fetoprotein (AFP), carcinoembryonicantigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1,mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumorprotein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1(PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72,HER2, GD2, cMET, EGFR, Mesothelin, VEGFR, alpha-folate receptor, C7R,IL-3, Cancer-testis antigen, MART-1 gp100, and TNF-relatedapoptosis-inducing ligand.

In certain embodiments, the tumor antigen is a carcinoembryonic antigen(CEA). In certain embodiments, the tumor antigen is an epithelial tumorantigen (ETA).

In certain embodiments, the tumor antigen is a mucin. In some of theseembodiments, the mucin is a secreted mucin. In some other of theseembodiments, the mucin is a transmembrane mucin. In specificembodiments, the tumor antigen is mucin 1 (MUC1). In specificembodiments, the tumor antigen is Tn-MUC1. In specific embodiments, thetumor antigen is mucin 16 (MUC16).

In certain embodiments, the tumor antigen is a melanoma-associatedantigen (MAGE). In some of these embodiments, the MAGE is a type-I MAGE.In some other of these embodiments, the MAGE is a type-II MAGE. Inspecific embodiments, the type-I MAGE is MAGE-A2. In specificembodiments, the type-I MAGE is MAGE-A4.

In certain embodiments, the tumor antigen is alpha-fetoprotein (AFP). Incertain embodiments, the tumor antigen is tumor protein p53 (p53). Incertain embodiments, the tumor antigen is tyrosinase. In certainembodiments, the tumor antigen is a tyrosinase-related protein (TRP). Insome embodiments, the tumor antigen is programmed death ligand 1 (PD-L1)or programmed death ligand 2 (PD-L2). In various embodiments, the tumorantigen is selected from the group consisting of CD4, CD8, CD45, CD80,and CD86.

In some embodiments, the immunomodulating component is a chimericantigen receptor (CAR) or a derivative thereof. In some embodiments, theCAR binds to one or more of alpha-fetoprotein (AFP), carcinoembryonicantigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1,mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumorprotein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1(PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72,HER2, GD2, cMET, EGFR, Mesothelin, VEGFR, alpha-folate receptor, C7R,IL-3, Cancer-testis antigen, MART-1 gp100, and TNF-relatedapoptosis-inducing ligand.

In some embodiments, the additional immunomodulating component is anactivator of a T-cell receptor or co-receptor. In certain embodiments,the additional immunomodulating component is an activator of CD3. Incertain embodiments, the activator is a fragment of a monoclonalantibody of CD3. In certain embodiments, the antibody fragment is ascFv, (scFv)₂, Fab, Fab′, and F(ab′)₂, F(ab1)₂, Fv, dAb, or Fd of amonoclonal antibody of CD3. In certain embodiments, the activator is ananobody, a bispecific antibody, or a multispecific antibody againstCD3. In certain embodiments, the additional immunomodulating componentis an activator of CD28. In certain embodiments, the activator is afragment of a monoclonal antibody of CD28. In certain embodiments, theantibody fragment is a scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂, F(ab1)₂,Fv, dAb, or Fd of a monoclonal antibody of CD28. In certain embodiments,the activator is a nanobody, a bispecific antibody, or a multispecificantibody against CD28.

In some embodiments, the additional immunomodulating component is amajor histocompatibility complex (MHC) or a derivative thereof. In someof these embodiments, the additional immunomodulating component is anMHC class I or a derivative thereof. In some of these embodiments, theadditional immunomodulating component is an MHC class II or a derivativethereof. In some of these embodiments, the additional immunomodulatingcomponent is an MHC class III or a derivative thereof.

In some embodiments, the additional immunomodulating component is ahuman leukocyte antigen (HLA) or a derivative thereof. In some of theseembodiments, the additional immunomodulating component is an HLA-A,HLA-B, HLA-C, or derivative thereof. In some of these embodiments, theadditional immunomodulating component is an HLA-E, HLA-F, HLA-G, or aderivative thereof. In some of these embodiments, the additionalimmunomodulating component is an HLA-DP, HLA-DQ, HLA-DR, or a derivativethereof.

In various embodiments, the immunomodulating component can be apolypeptide, a polynucleotide, a polysaccharide, a lipid, a smallmolecule, or a toxin.

In some embodiments, the immunomodulating component can be a protein, apeptide, a glycolipid, or a glycoprotein.

In certain embodiments, the immunomodulating component is an agonist. Insome of these embodiments, the agonist is an endogenous agonist, such asa hormone, or a neurotransmitter. In some other of these embodiments,the agonist is an exogenous agonist, such as a drug. In someembodiments, the agonist is a physical agonist, which can create anagonist response without binding to the receptor. In some embodiments,the agonist is a superagonist, which can produce a greater maximalresponse than the endogenous agonist. In certain embodiments, theagonist is a full agonist with full efficacy at the receptor. In certainother embodiments, the agonist is a partial agonist having only partialefficacy at the receptor relative to a full agonist. In someembodiments, the agonist is an inverse agonist that can inhibit theconstitutive activity of the receptor. In some embodiments, the agonistis a co-agonist that works with other co-agonists to produce an effecton the receptor. In certain embodiments, the agonist is an irreversibleagonist that binds permanently to a receptor through formation ofcovalent bond. In certain embodiments, the agonist is selective agonistfor a specific type of receptor.

In certain embodiments, the immunomodulating component is an antagonist.In some of these embodiments, the antagonist is a competitiveantagonist, which reversibly binds to the receptor at the same bindingsite as the endogenous ligand or agonist without activating thereceptor. Competitive antagonist can affect the amount of agonistnecessary to achieve a maximal response. In some other of theseembodiments, the antagonist is a non-competitive antagonist, which bindsto an active site of the receptor or an allosteric site of the receptor.Non-competitive antagonist can reduce the magnitude of the maximumresponse that can be attained by any amount of agonist. In some otherembodiments, the antagonist is an uncompetitive antagonist, whichrequires receptor activation by an agonist before its binding to aseparate allosteric binding site.

In various embodiments, the immunomodulating component comprises anantibody or an antigen-binding fragment. The immunomodulating componentcan be a full length protein or a fragment thereof. The antibody orantigen-binding fragment can be derived from natural sources, or partlyor wholly synthetically produced. In some embodiments, the antibody is amonoclonal antibody. In some of these embodiments, the monoclonalantibody is an IgG antibody. In certain embodiments, the monoclonalantibody is an IgG1, IgG2, IgG3, or IgG4. In some other embodiments, theantibody is a polyclonal antibody. In certain embodiments, theantigen-binding fragment is selected from Fab, Fab′, and F(ab′)₂,F(ab1)₂, Fv, dAb, and Fd fragments. In certain embodiments, theantigen-binding fragment is an scFv or (scFv)₂ fragment. In certainother embodiments, the antibody or antigen-binding fragment is aNanobody® (single-domain antibody). In some embodiments, the antibody orantigen-binding fragment is a bispecific or multispecific antibody.

In various embodiments, the antibody or antigen-binding fragment isfully human. In some embodiments, the antibody or antigen-bindingfragment is humanized. In some embodiments, the antibody orantigen-binding fragment is chimeric. In some of these embodiments, thechimeric antibody has non-human V region domains and human C regiondomains. In some embodiments, the antibody or antigen-binding fragmentis non-human, such as murine or veterinary.

In some embodiments, the immunomodulating component is a protein, apeptide, a glycolipid, or a glycoprotein.

In various embodiments, the composition comprises two or more abovementioned immunomodulating components, including mixtures, fusions,combinations and conjugates, of atoms, molecules, etc. In certainembodiments, the composition comprises a nucleic acid combined with apolypeptide. In certain embodiments, the composition comprises two ormore polypeptides conjugated to each other. In certain embodiments, thecomposition comprises a protein conjugated to a biologically activemolecule. In some of these embodiments, the biologically active moleculeis a prodrug.

The Pharmaceutical Composition

The pharmaceutical compositions generally comprise a plurality ofextracellular vesicles and a pharmaceutically-acceptable excipient orcarrier in a form suitable for administration to a subject.Pharmaceutically-acceptable excipients or carriers are determined inpart by the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions comprising a plurality of extracellular vesicles. (See,e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 21st ed. (2005)). The pharmaceutical compositions are generallyformulated sterile and in full compliance with all Good ManufacturingPractice (GMP) regulations of the U.S. Food and Drug Administration.

In some embodiments, the pharmaceutical composition comprises one ormore therapeutic agents and the extracellular vesicle described herein.In some embodiments, the extracellular vesicles are co-administered withof one or more separate therapeutic agents, wherein co-administrationincludes administration of the separate therapeutic agent before, afteror concurrent with administration of the extracellular vesicles.

Pharmaceutically-acceptable excipients include excipients that aregenerally safe, non-toxic, and desirable, including excipients that areacceptable for veterinary use as well as for human pharmaceutical use.

Examples of carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and compounds for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or compound is incompatible with the extracellular vesiclesdescribed herein, use thereof in the compositions is contemplated.Supplementary therapeutic agents can also be incorporated into thecompositions. Typically, a pharmaceutical composition is formulated tobe compatible with its intended route of administration. Theextracellular vesicles can be administered by parenteral, topical,intravenous, oral, subcutaneous, intra-arterial, intradermal,transdermal, rectal, intracranial, intraperitoneal, intranasal,intramuscular route or as inhalants. In certain embodiments, thepharmaceutical composition comprising extracellular vesicles isadministered intravenously, e.g., by injection. The extracellularvesicles can optionally be administered in combination with othertherapeutic agents that are at least partly effective in treating thedisease, disorder or condition for which the extracellular vesicles areintended.

Solutions or suspensions can include the following components: a sterilediluent such as water, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial compounds such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelatingcompounds such as ethylenediaminetetraacetic acid (EDTA); buffers suchas acetates, citrates or phosphates, and compounds for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Thepreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (if water soluble) or dispersions and sterile powders.For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The composition is generally sterileand fluid to the extent that easy syringeability exists. The carrier canbe a solvent or dispersion medium containing, e.g., water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, e.g., by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalcompounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. If desired, isotonic compounds, e.g., sugars,polyalcohols such as manitol, sorbitol, and sodium chloride can be addedto the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition a compound whichdelays absorption, e.g., aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theextracellular vesicles in an effective amount and in an appropriatesolvent with one or a combination of ingredients enumerated herein, asdesired. Generally, dispersions are prepared by incorporating theextracellular vesicles into a sterile vehicle that contains a basicdispersion medium and any desired other ingredients. In the case ofsterile powders for the preparation of sterile injectable solutions,methods of preparation are vacuum drying and freeze-drying that yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The extracellularvesicles can be administered in the form of a depot injection or implantpreparation which can be formulated in such a manner to permit asustained or pulsatile release of the extracellular vesicles.

Systemic administration of compositions comprising extracellularvesicles can also be by transmucosal means. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, e.g., for transmucosal administration, detergents,bile salts, and fusidic acid derivatives. Transmucosal administrationcan be accomplished through the use of, e.g., nasal sprays.

In certain embodiments the pharmaceutical composition comprisingextracellular vesicles is administered intravenously into a subject thatwould benefit from the pharmaceutical composition. In certain otherembodiments, the composition is administered to the lymphatic system,e.g., by intralymphatic injection or by intranodal injection (see e.g.,Senti et al., PNAS 105(46): 17908 (2008)), or by intramuscularinjection, by subcutaneous administration, by direct injection into thethymus, or into the liver.

In certain embodiments, the pharmaceutical composition comprisingextracellular vesicles is administered as a liquid suspension. Incertain embodiments, the pharmaceutical composition is administered as aformulation that is capable of forming a depot following administration.In certain preferred embodiments, the depot slowly releases theextracellular vesicles into circulation, or remains in depot form.

Typically, pharmaceutically-acceptable compositions are highly purifiedto be free of contaminants, are biocompatible and not toxic, and aresuited to administration to a subject. If water is a constituent of thecarrier, the water is highly purified and processed to be free ofcontaminants, e.g., endotoxins.

The pharmaceutically-acceptable carrier can be lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate,alginates, gelatin, calcium silicate, micro-crystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and/or mineral oil, but is not limited thereto. Thepharmaceutical composition can further include a lubricant, a wettingagent, a sweetener, a flavor enhancer, an emulsifying agent, asuspension agent, and/or a preservative.

The pharmaceutical compositions described herein comprise theextracellular vesicles described herein and optionally apharmaceutically active or therapeutic agent. The therapeutic agent canbe a biological agent, a small molecule agent, or a nucleic acid agent.

Dosage forms are provided that comprise a pharmaceutical compositioncomprising the extracellular vesicles described herein. In someembodiments, the dosage form is formulated as a liquid suspension forintravenous injection.

In certain embodiments, the preparation of extracellular vesicles issubjected to radiation, e.g., X rays, gamma rays, beta particles, alphaparticles, neutrons, protons, elemental nuclei, UV rays in order todamage residual replication-competent nucleic acids.

In certain embodiments, the preparation of extracellular vesicles issubjected to gamma irradiation using an irradiation dose of more than 1,5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100kGy.

In certain embodiments, the preparation of extracellular vesicles issubjected to X-ray irradiation using an irradiation dose of more than0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.

Methods

Aspects of the subject disclosure also include methods of producing thecomposition comprising the extracellular vesicle and theimmunomodulating component. In some embodiments, the method comprises:obtaining the extracellular vesicle from the producer cell, wherein theproducer cell naturally contains the immunomodulating component; andoptionally isolating the obtained extracellular vesicle. In someembodiments, the method comprises: modifying a producer cell with theimmunomodulating component; obtaining the extracellular vesicle from themodified producer cell; and optionally isolating the obtainedextracellular vesicles. In some other embodiments, the method comprises:obtaining the extracellular vesicle from a producer cell; isolating theobtained extracellular vesicles; and modifying the isolatedextracellular vesicle with the immunomodulating component. In certainembodiments, the method further comprises formulating the isolatedextracellular vesicles into a pharmaceutical composition.

Methods of Producing the Extracellular Vesicles

Methods of Modifying the Producer Cell with the ImmunomodulatingComponent

In various embodiments, the method comprises modifying a producer cellwith the immunomodulating component.

The producer cell can be a mammalian cell line, a plant cell line, aninsect cell line, a fungi cell line, or a prokaryotic cell line. Incertain embodiments, the producer cell is a mammalian cell line. Themammalian cell lines include but are not limited to a human embryonickidney (HEK) cell line, a Chinese hamster ovary (CHO) cell line, anHT-1080 cell line, a HeLa cell line, a PERC-6 cell line, a CEVEC cellline, a fibroblast cell line, an amniocyte cell line, an epithelial cellline, and a mesenchymal stem cell (MSC) cell line. In some preferredembodiments, the mammalian cell line can be HEK-293 cells, BJ humanforeskin fibroblast cells, fHDF fibroblast cells, AGE.HN® neuronalprecursor cells, CAP® amniocyte cells, adipose mesenchymal stem cells,or RPTEC/TERT1 cells. The producer cell can also be a primary cell. Invarious embodiments, the primary cell can be a primary mammalian cell, aprimary plant cell, a primary insect cell, a primary fungi cell, or aprimary prokaryotic cell.

In certain preferred embodiments, the producer cell is an immune cell,such as a dendritic cell, a T cell, a B cell, a natural killer cell (NKcell), an antigen presenting cell, a macrophage, a T helper cell, or aregulatory T cell (Treg cell).

In various embodiments, the immunomodulating component can be expressedin a producer cell from a transgene or mRNA introduced into the producercell by transfection (see, e.g., Bacchetti S, Graham F L (April 1977).“Transfer of the gene for thymidine kinase to thymidine kinase-deficienthuman cells by purified herpes simplex viral DNA”. Proceedings of theNational Academy of Sciences of the United States of America. 74 (4):1590-4; Kriegler M (1991). Transfer and Expression: A Laboratory Manual.W. H. Freeman. pp. 96-97; Felgner P L, Gadek T R, Holm M, Roman R, ChanH W, Wenz M, Northrop J P, Ringold G M, Danielsen M (November 1987).“Lipofection: a highly efficient, lipid-mediated DNA-transfectionprocedure”. Proceedings of the National Academy of Sciences of theUnited States of America. 84 (21): 7413-7; Felgner J H, Kumar R, SridharC N, Wheeler C J, Tsai Y J, Border R, Ramsey P, Martin M, Felgner P L(January 1994). “Enhanced gene delivery and mechanism studies with anovel series of cationic lipid formulations”. The Journal of BiologicalChemistry. 269 (4): 2550-61), viral transduction (see, e.g., Griffiths AJ, Miller J H, Suzuki D T, Lewontin R C, Gelbart W M (2000).“Tansducton”. An Introduction to Genetic Analysis (7th ed.)),electroporation (see, e.g., Neumann, E; Schaefer-Ridder, M; Wang, Y;Hofschneider, P H (1982). “Gene transfer into mouse lyoma cells byelectroporation in high electric fields”. The EMBO Journal. 1 (7):841-5)) extrusion (see, e.g., Sharei A, Zoldan J, Adamo A, Sim W Y, ChoN, Jackson E, Mao S, Schneider S, Han M J, Lytton-Jean A, Basto P A,Jhunjhunwala S, Lee J, Heller D A, Kang J W, Hartoularos G C, Kim K S,Anderson D G, Langer R, Jensen K F (February 2013). “A vector-freemicrofluidic platform for intracellular delivery”. Proceedings of theNational Academy of Sciences of the United States of America. 110 (6):2082-7), sonication (see, e.g., Yizhi Song (2007). “Ultrasound-mediatedDNA transfer for bacteria”. Nucleic Acids Res. 35 (19): e129.), cellfusion (see, e.g., Felgner P L, Gadek T R, Holm M, Roman R, Chan H W,Wenz M, Northrop J P, Ringold G M, Danielsen M (November 1987).“Lipofection: a highly efficient, lipid-mediated DNA-transfectionprocedure”. Proceedings of the National Academy of Sciences of theUnited States of America. 84 (21): 7413-), or other methods that areknown to the skilled in the art.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by transfection. In some embodiments, theimmunomodulating component can be introduced into suitable producercells using synthetic macromolecules such as cationic lipids andpolymers (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). In someembodiments, the cationic lipids form complexes with theimmunomodulating component through charge interactions. In some of theseembodiments, the positively charged complexes bind to the negativelycharged cell surface and are taken up by the cell by endocytosis. Insome other embodiments, a cationic polymer can be used to transfectproducer cells. In some of these embodiments, the cationic polymer ispolyethylenimine (PEI). In certain embodiments, chemicals such ascalcium phosphate, cyclodextrin, or polybrene, can be used to introducethe immunomodulating component to the producer cells. Theimmunomodulating component can also be introduced into a producer cellusing a physical method such as particle-mediated transfection, “genegun,” biolistics, or particle bombardment technology (Papapetrou et al.,Gene Therapy 12: S118-S130 (2005)). A reporter gene such as, forexample, beta-galactosidase, chloramphenicol acetyltransferase,luciferase, or green fluorescent protein can be used to assess thetransfection efficiency of the producer cell.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by viral transduction. A number of viruses can be usedas gene transfer vehicles, including moloney murine leukemia virus(MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus(HSV), lentiviruses, and spumaviruses. The viral mediated gene transfervehicles comprise vectors based on DNA viruses, such as adenovirus,adeno-associated virus and herpes virus, as well as retroviral basedvectors.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by electroporation. Electroporation creates transientpores in the cell membrane, allowing for the introduction of variousmolecules into the cell. In some embodiments, DNA and RNA as well aspolypeptides and non-polypeptide therapeutic agents can be introducedinto the producer cell by electroporation.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by microinjection. In some embodiments, a glassmicropipette can be used to inject the immunomodulating component intothe producer cell at the microscopic level.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by extrusion.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by sonication. In some embodiments, the producer cellis exposed to high intensity sound waves, causing transient disruptionof the cell membrane allowing loading of an immunomodulating component.

In certain embodiments, the immunomodulating component is introduced tothe producer cell by cell fusion. In some embodiments, theimmunomodulating component is introduced by electrical cell fusion. Insome other embodiments, polyethylene glycol (PEG) is used to fuse theproducer cells. In some other embodiments, sendai virus is used to fusethe producer cells.

In some embodiments, the immunomodulating component is introduced to theproducer cell by hypotonic lysis. In some of these embodiments, theproducer cell is exposed to low ionic strength buffer causing them toburst allowing loading of an immunomodulating component. In somealternative embodiments, controlled dialysis against a hypotonicsolution is used to swell the producer cell and to create pores in theproducer cell membrane. The producer cell is subsequently exposed toconditions that allow resealing of the membrane.

In some embodiments, the immunomodulating component is introduced to theproducer cell by detergent treatment. In certain embodiments, producercell is treated with a mild detergent which transiently compromises theproducer cell membrane by creating pores allowing loading of animmunomodulating component. After producer cells are loaded, thedetergent is washed away thereby resealing the membrane.

In some embodiments, the immunomodulating component is introduced to theproducer cell by receptor mediated endocytosis. In certain embodiments,producer cells have a surface receptor which upon binding of theimmunomodulating component induces internalization of the receptor andthe associated immunomodulating component.

In some embodiments, the immunomodulating component is introduced to theproducer cell by filtration. In certain embodiments, the producer cellsand the immunomodulating component can be forced through a filter ofpore size smaller than the producer cell causing transient disruption ofthe producer cell membrane and allowing the immunomodulating componentto enter the producer cell.

In some embodiments, the producer cell is subjected to several freezethaw cycles, resulting in cell membrane disruption allowing loading ofan immunomodulating component.

Methods of Modifying the Extracellular Vesicle with the ImmunomodulatingComponent

In various alternative embodiments, the immunomodulating component isintroduced directly to the extracellular vesicles after the isolation ofthe extracellular vesicles.

In certain embodiments, the immunomodulating component is introduced tothe extracellular vesicle by transfection. In some embodiments, theimmunomodulating component can be introduced into the extracellularvesicles using synthetic macromolecules such as cationic lipids andpolymers (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). Incertain embodiments, chemicals such as calcium phosphate, cyclodextrin,or polybrene, can be used to introduce the immunomodulating component tothe extracellular vesicles.

In certain embodiments, the immunomodulating component is introduced tothe extracellular vesicle by electroporation. In some embodiments,extracellular vesicles are exposed to an electrical field which causestransient holes in the extracellular vesicle membrane, allowing loadingof an immunomodulating component.

In certain embodiments, the immunomodulating component is introduced tothe extracellular vesicle by microinjection. In some embodiments, aglass micropipette can be used to inject the immunomodulating componentdirectly into the extracellular vesicle at the microscopic level.

In certain embodiments, the immunomodulating component is introduced tothe extracellular vesicle by extrusion.

In certain embodiments, the immunomodulating component is introduced tothe extracellular vesicle by sonication. In some embodiments,extracellular vesicles are exposed to high intensity sound waves,causing transient disruption of the extracellular vesicle membraneallowing loading of an immunomodulating component.

In some embodiments, the immunomodulating component can be conjugated tothe surface of the extracellular vesicle. Conjugation can be achievedchemically or enzymatically, by methods known in the art.

In some embodiments, the extracellular vesicle comprises animmunomodulating component that is chemically conjugated. Chemicalconjugation can be accomplished by covalent bonding of theimmunomodulating component to another molecule, with or without use of alinker. The formation of such conjugates is within the skill of artisansand various techniques are known for accomplishing the conjugation, withthe choice of the particular technique being guided by the materials tobe conjugated. In certain embodiments, polypeptides are conjugated tothe extracellular vesicle. In certain other embodiments,non-polypeptides, such as lipids, carbohydrates, nucleic acids, andsmall molecules, are conjugated to the extracellular vesicle.

In some embodiments, the immunomodulating component is introduced to theextracellular vesicle by hypotonic lysis. In some of these embodiments,the extracellular vesicles are exposed to low ionic strength buffercausing them to burst allowing loading of an immunomodulating component.In some alternative embodiments, controlled dialysis against a hypotonicsolution is used to swell the extracellular vesicle and to create poresin the extracellular vesicle membrane. The extracellular vesicle issubsequently exposed to conditions that allow resealing of the membrane.

In some embodiments, the immunomodulating component is introduced to theextracellular vesicle by detergent treatment. In certain embodiments,extracellular vesicles are treated with a mild detergent whichtransiently compromises the extracellular vesicle membrane by creatingpores allowing loading of an immunomodulating component. Afterextracellular vesicles are loaded, the detergent is washed away therebyresealing the membrane.

In some embodiments, the immunomodulating component is introduced to theextracellular vesicle by receptor mediated endocytosis. In certainembodiments, extracellular vesicles have a surface receptor which uponbinding of the immunomodulating component induces internalization of thereceptor and the associated immunomodulating component.

In some embodiments, the immunomodulating component is introduced to theextracellular vesicle by mechanical firing. In certain embodiments,extracellular vesicles can be bombarded with an immunomodulatingcomponent attached to a heavy or charged particle such as goldmicrocarriers. In some of these embodiments, the particle can bemechanically or electrically accelerated such that it traverses theextracellular vesicle membrane.

In some embodiments, the immunomodulating component is introduced to theextracellular vesicle by filtration. In certain embodiments, theextracellular vesicles and the immunomodulating component can be forcedthrough a filter of pore size smaller than the extracellular vesiclecausing transient disruption of the extracellular vesicle membrane andallowing the immunomodulating component to enter the extracellularvesicle.

In some embodiments, extracellular vesicles are subjected to severalfreeze thaw cycles, resulting in extracellular vesicle membranedisruption allowing loading of an immunomodulating component.

Methods of Isolating the Extracellular Vesicles

The extracellular vesicles can be isolated from the producer cells. Incertain embodiments, the extracellular vesicle is released by theproducer cell into the cell culture medium. It is contemplated that allknown manners of isolation of extracellular vesicles are deemed suitablefor use herein. For example, physical properties of extracellularvesicles can be employed to separate them from a medium or other sourcematerial, including separation on the basis of electrical charge (e.g.,electrophoretic separation), size (e.g., filtration, molecular sieving,etc.), density (e.g., regular or gradient centrifugation), Svedbergconstant (e.g., sedimentation with or without external force, etc.).Alternatively, or additionally, isolation can be based on one or morebiological properties, and include methods that can employ surfacemarkers (e.g., for precipitation, reversible binding to solid phase,FACS separation, specific ligand binding, non-specific ligand binding,affinity purification etc.).

Isolation and enrichment can be done in a general and non-selectivemanner, typically including serial centrifugation. Alternatively,isolation and enrichment can be done in a more specific and selectivemanner, such as using extracellular vesicle or producer cell-specificsurface markers. For example, specific surface markers can be used inimmunoprecipitation, FACS sorting, affinity purification, and magneticseparation with bead-bound ligands.

In some embodiments, size exclusion chromatography can be utilized toisolate the extracellular vesicles. Size exclusion chromatographytechniques are known in the art. Exemplary, non-limiting techniques areprovided herein. In some embodiments, a void volume fraction is isolatedand comprises the extracellular vesicles of interest. Further, in someembodiments, the extracellular vesicles can be further isolated afterchromatographic separation by centrifugation techniques (of one or morechromatography fractions), as is generally known in the art. In someembodiments, for example, density gradient centrifugation can beutilized to further isolate the extracellular vesicles. In certainembodiments, it can be desirable to further separate the producercell-derived extracellular vesicles from extracellular vesicles of otherorigin. For example, the producer cell-derived extracellular vesiclescan be separated from non-producer cell-derived extracellular vesiclesby immunosorbent capture using an antigen antibody specific for theproducer cell.

In some embodiments, the isolation of extracellular vesicles can involvecombinations of methods that include, but are not limited to,differential centrifugation, size-based membrane filtration,immunoprecipitation, FACS sorting, and magnetic separation.

Methods of Measuring the Size of Extracellular Vesicles

In some embodiments, the methods described herein comprise measuring thesize of extracellular vesicles and/or populations of extracellularvesicles. Generally, extracellular vesicle size is measured as thelongest measurable dimension. Generally, the longest measurabledimension of an extracellular vesicle is also referred to as itsdiameter.

Extracellular vesicle size can be measured using dynamic lightscattering (DLS) and/or multiangle light scattering (MALS). Methods ofusing DLS and/or MALS to measure the size of extracellular vesicles areknown to those of skill in the art, and include the nanoparticletracking assay (NTA, e.g., using a Malvern NanoSight NS300 nanoparticletracking device). In a specific embodiment, the extracellular vesiclesize is determined using a Malvern NanoSight NS300. In some embodiments,the extracellular vesicles described herein have a longest dimension ofabout 20-300 nm as measured by NTA (e.g., using a Malvern NanoSightNS300). In other embodiments, the extracellular vesicles describedherein have a longest dimension of about 40-200 nm as measured by NTA(e.g., using a Malvern NanoSight NS300). In other embodiments, theextracellular vesicle populations described herein comprise apopulation, wherein 90% of the extracellular vesicles have a longestdimension of about 20-300 nm as measured by NTA (e.g., using a MalvernNanoSight NS300). In other embodiments, the extracellular vesiclepopulations described herein comprise a population, wherein 95% of theextracellular vesicles have a longest dimension of about 20-300 nm asmeasured by NTA (e.g., using a Malvern NanoSight NS300). In otherembodiments, the extracellular vesicle populations described hereincomprise a population, wherein 99% of the extracellular vesicles have alongest dimension of about 20-300 nm as measured by NTA (e.g., using aMalvern NanoSight NS300). In other embodiments, the extracellularvesicle populations described herein comprise a population, wherein 90%of the extracellular vesicles have a longest dimension of about 40-200nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In otherembodiments, the extracellular vesicle populations described hereincomprise a population, wherein 95% of the extracellular vesicles have alongest dimension of about 40-200 nm as measured by NTA (e.g., using aMalvern NanoSight NS300). In other embodiments, the extracellularvesicle populations described herein comprise a population, wherein 99%of the extracellular vesicles have a longest dimension of about 40-200nm as measured by NTA (e.g., using a Malvern NanoSight NS300).

Extracellular vesicle size can be measured using tunable resistive pulsesensing (TRPS). In a specific embodiment, extracellular vesicle size asmeasured by TRPS is determined using an iZON qNANO Gold. In someembodiments, the extracellular vesicles described herein have a longestdimension of about 20-300 nm as measured by TRPS (e.g., using an iZONqNano Gold). In other embodiments, the extracellular vesicles describedherein have a longest dimension of about 40-200 nm as measured by TRPS(e.g., an iZON qNano Gold). In other embodiments, the extracellularvesicle populations described herein comprise a population, wherein 90%of the extracellular vesicles have a longest dimension of about 20-300nm as measured by TRPS (e.g., using an iZON qNano Gold). In otherembodiments, the extracellular vesicle populations described hereincomprise a population, wherein 95% of the extracellular vesicles have alongest dimension of about 20-300 nm as measured by TRPS (e.g., using aniZON qNano Gold). In other embodiments, the extracellular vesiclepopulations described herein comprise a population, wherein 99% of theextracellular vesicles have a longest dimension of about 20-300 nm asmeasured by TRPS (e.g., using an iZON qNano Gold). In other embodiments,the extracellular vesicle populations described herein comprise apopulation, wherein 90% of the extracellular vesicles have a longestdimension of about 40-200 nm as measured by TRPS (e.g., using an iZONqNano Gold). In other embodiments, the extracellular vesicle populationsdescribed herein comprise a population, wherein 95% of the extracellularvesicles have a longest dimension of about 40-200 nm as measured by TRPS(e.g., using an iZON qNano Gold). In other embodiments, theextracellular vesicle populations described herein comprise apopulation, wherein 99% of the extracellular vesicles have a longestdimension of about 40-200 nm as measured by TRPS (e.g., using an iZONqNano Gold).

Extracellular vesicles size can be measured using electron microscopy.In some embodiments, the method of electron microscopy used to measureextracellular vesicle size is transmission electron microscopy. In aspecific embodiment, the transmission electron microscope used tomeasure extracellular vesicle size is a Tecnai™ G² Spirit BioTWIN.Methods of measuring extracellular vesicle size using an electronmicroscope are well-known to those of skill in the art, and any suchmethod can be appropriate for measuring extracellular vesicle size. Insome embodiments, the extracellular vesicles described herein have alongest dimension of about 20-300 nm as measured by a scanning electronmicroscope (e.g., a Tecnai™ G² Spirit BioTWIN scanning electronmicroscope). In other embodiments, the extracellular vesicles describedherein have a longest dimension of about 40-200 nm as measured by ascanning electron microscope (e.g., a Tecnai™ G² Spirit BioTWIN scanningelectron microscope). In other embodiments, the extracellular vesiclepopulations described herein comprise a population, wherein 90% of theextracellular vesicles have a longest dimension of about 20-300 nm asmeasured by a scanning electron microscope (e.g., a Tecnai™ G² SpiritBioTWIN scanning electron microscope). In other embodiments, theextracellular vesicle populations described herein comprise apopulation, wherein 95% of the extracellular vesicles have a longestdimension of about 20-300 nm as measured by a scanning electronmicroscope (e.g., a Tecnai™ G² Spirit BioTWIN scanning electronmicroscope). In other embodiments, the extracellular vesicle populationsdescribed herein comprise a population, wherein 99% of the extracellularvesicles have a longest dimension of about 20-300 nm as measured by ascanning electron microscope (e.g., a Tecnai™ G² Spirit BioTWIN scanningelectron microscope). In other embodiments, the extracellular vesiclepopulations described herein comprise a population wherein 90% of theextracellular vesicles have a longest dimension of about 40-200 nm asmeasured by a scanning electron microscope (e.g., a Tecnai™ G² SpiritBioTWIN scanning electron microscope). In other embodiments, theextracellular vesicle populations described herein comprise a populationwherein 95% of the extracellular vesicles have a longest dimension ofabout 40-200 nm as measured by a scanning electron microscope (e.g., aTecnai™ G² Spirit BioTWIN scanning electron microscope). In otherembodiments, the extracellular vesicle populations described hereincomprise a population wherein 99% of the extracellular vesicles have alongest dimension of about 40-200 nm as measured by a scanning electronmicroscope (e.g., a Tecnai™ G² Spirit BioTWIN scanning electronmicroscope).

Methods for Determining Macrophage Polarization

Disclosed herein are methods and compositions for increasing macrophagepolarization from an M2 to an M1 phenotype using an extracellularvesicle comprising one or more immunomodulating component(s) thatinhibit expression of a macrophage target gene. The compositions arepreferentially taken up by macrophages (as compared to other cell typessuch as T-cells, B-cells, macrophages, or dendritic cells), and are asor more effective in increasing macrophage polarization than anequimolar amount of the immunomodulating component alone. Methods fordetermining macrophage polarization, including detection of M2 and M1phenotypes of macrophages, can be performed by any method known in theart for the determination of M2 and M1 phenotypes. Tissue sections(e.g., from tumor biopsies), blood samples, etc. can be assayed (e.g.,stained) for markers of pan-macrophages as well as M2 and M1 macrophagesincluding, but not limited to, M2 cell surface markers (e.g., YM1,FIZZ1, Dectin-1, MGL), M2 associated cytokines (e.g., IL-10, TGFβ, PGE2,CCL2, CCL17, CCL18, CCL22 and CCL24), M1 associated cytokines (e.g.,INFγ, IL-12, IL-23, TNFα, IL-6, IL-1, CCL5, CSCL9, CXCL10 and CXCL11),growth factors (e.g., VEGF-A, VEGF-C, EGF, and TGF-β), enzymes (e.g.,matrix metalloproteinases MMP2, MMP9, cysteine cathepsin proteases); M2associated miRNAs (e.g., miRNA146a, miRNA let 7b, and miR-223) and/or M1associated miRNAs (e.g., miRNA155, miR-33). See, e.g., Mosser, D. M., &Edwards, J. P. (2008). Exploring the full spectrum of macrophageactivation. Nature Reviews Immunology, 8(12), 958-969; Murray, P. J.,Allen, J. E., Biswas, S. K., Fisher, E. A., Gilroy, D. W., Goerdt, S.,Wynn, T. A. (2014). Macrophage activation and polarization: nomenclatureand experimental guidelines. Immunity, 41(1), 14-20. Macrophages and/ormacrophage components, secretions or macrophage activity within samples(see, e.g., Gautier, E. L., Shay, T., Miller, J., Greter, M., Jakubzick,C., Ivanov, S., Randolph, G. J. (2007). Gene expression profiles andtranscriptional regulatory pathways underlying mouse tissue macrophageidentity and diversity, Nature Immunology 13(11), 1118-1128) can bedetected by flow cytometry, immunohisto-chemistry, immunoblotting,quantitative PCR, or any other method known in the art to detect cells,or cellular products in biological samples.

Methods of Treating Cancer

Also, provided herein are methods of treating cancer ina subject.

In various embodiments, the composition of extracellular vesicles, e.g.,exosomes, comprising one or more immunomodulating components thatinhibit at least one gene and thereby increase macrophage polarizationfrom the M2 to M1 phenotype is administered to a subject with cancer. Insome of these embodiments, the composition can up-regulate an immuneresponse and enhance the tumor targeting of the subject's immune system.In some aspects, the increased macrophage polarization from the M2 to M1phenotype per se up-regulates the subject's immune response and enhancesthe tumor targeting of the subject's immune system. Some authors mentionthe M2d subtype activation as a response to IL-6 and adenosines, andthese macrophages are also referred as tumor-associated macrophages(TAM). See, e.g., Röszer, T. (2015). Understanding the Mysterious M2Macrophage through Activation Markers and Effector Mechanisms. Mediatorsof Inflammation, 2015, 1-16; Funes, S. C., Rios, M., Escobar-Vera, J., &Kalergis, A. M. (2018). Implications of macrophage polarization inautoimmunity. Immunology, 154(2), 186-195; and Q. Wang, H. Ni, L. Lan,X. Wei, R. Xiang, and Y. Wang, “Fra-1 protooncogene regulates IL6expression in macrophages and promotes the generation of M2dmacrophages,” Cell Research, vol. 20, no. 6, pp. 701-712, 2010.Tumor-associated macrophages (TAM) are typical for their protumoralfunctions like promotion of cancer cell motility, metastasis formationand angiogenesis and their formation is dependent on microenvironmentalfactors which are present in developing tumor. TAMs produceimmunosuppressive cytokines like IL-10, TGFβ, PGE2 and a very smallamount of NO or ROI and low levels of inflammatory cytokines (IL-12,IL-10, TNFα, IL-6). Ability of TAMs to present tumor-associated antigensis decreased as well as stimulation of the anti-tumor functions of T andNK cells. Also TAMs are not able to lyse tumor cells.https://en.wikipedia.org/wiki/Macrophage_polarization-cite_note-Sica2008-31Targeting of TAM may be a novel therapeutic strategy against cancer, ashas been demonstrated through the delivery of agents to either alter therecruitment and distribution of TAMs, deplete existing TAMs, or inducethe re-education of TAMs from an M2 to an M1 phenotype. See, e.g.,Lewis, Claire E., and Jeffrey W. Pollard. “Distinct role of macrophagesin different tumor microenvironments.” Cancer research 66.2 (2006):605-612; Sica, Antonio, et al. “Macrophage polarization in tumourprogression.” Seminars in Cancer Biology. Vol. 18. No. 5. AcademicPress, 2008; Sica, Antonio, et al. Autocrine production of IL-10mediates defective IL-12 production and NF-kappa B activation intumor-associated macrophages. J Immunol. 2000 Jan. 15; 164(2):762-7;Cuccarese, Michael F.; Dubach, J. Matthew; Pfirschke, Christina;Engblom, Camilla; Garris, Christopher; Miller, Miles A.; Pittet, MikaelJ.; Weissleder, Ralph (2017-02-08). “Heterogeneity of macrophageinfiltration and therapeutic response in lung carcinoma revealed by 3Dorgan imaging”. Nature Communications. 8: 14293; Zeisberger, S M;Odermatt, B; Marty, C; Zehnder-Fjallman, A H M; Ballmer-Hofer, K;Schwendener, R A (2006-07-11). “Clodronate-liposome-mediated depletionof tumour-associated macrophages: a new and highly effectiveantiangiogenic therapy approach”. British Journal of Cancer. 95 (3):272-281; Rodell, Christopher B.; Arlauckas, Sean P.; Cuccarese, MichaelF.; Garris, Christopher S.; Li, Ran; Ahmed, Maaz S.; Kohler, Rainer H.;Pittet, Mikael J.; Weissleder, Ralph (2018-05-21).“TLR7/8-agonist-loaded nanoparticles promote the polarization oftumour-associated macrophages to enhance cancer immunotherapy”. NatureBiomedical Engineering; Guerriero, Jennifer L.; Sotayo, Alaba;Ponichtera, Holly E.; Castrillon, Jessica A.; Pourzia, Alexandra L.;Schad, Sara; Johnson, Shawn F.; Carrasco, Ruben D.; Lazo, Suzan (March2017). “Class IIa HDAC inhibition reduces breast tumours and metastasesthrough anti-tumour macrophages”. Nature. 543 (7645): 428-432.

In some embodiments, the cancer being treated is characterized byinfiltration of leukocytes (T-cells, B-cells, macrophages, dendriticcells, monocytes) into the tumor microenvironment, or so-called “hottumors” or “inflammatory tumors.”. In some embodiments, the cancer beingtreated is characterized by low levels or undetectable levels ofleukocyte infiltration into the tumor microenvironment, or so-called“cold tumors” or “non-inflammatory tumors”. In some embodiments, thecomposition is administered in an amount and for a time sufficient toconvert a “cold tumor” into a “hot tumor”, i.e., said administeringresults in the infiltration of leukocytes (such as T-cells) into thetumor microenvironment.

In some embodiments, the compositions comprise an extracellular vesicleand a combination of more than one immunomodulating component, includinga component that promotes macrophage polarization from an M2 to an M1phenotype, and a component that additionally enhances an immuneresponse, such as a checkpoint blockade inhibitor, e.g., Ipilimumab,targeting CTLA-4, Nivolumab Cemiplimab and Pembrolizumab targeting PD-1,and Atezolizumab, Avelumab, Durvalumab, each targeting PD-L1, andinhibitors of CSFR-1, such as Pexidartinib, PLX7486, ARRY-382,JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab.Administration of these compositions as treatments for subjects withcancer can further up-regulate an immune response and enhance the tumortargeting of the subject's immune system through the combined actions ofthe immunomodulating components.

In some embodiments, the additional immunomodulating component is anantibody or active fragment that targets CTLA-4, PD-1, PD-L1, or CSF1-R.In embodiments, the antibody or active fragment thereof comprises CDRsthat are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the CDRs of Ipilimumab. In embodiments, the antibodyor active fragment thereof comprises CDRs that are at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDRs ofNivolumab. In embodiments, the antibody or active fragment thereofcomprises CDRs that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the CDRs of Cemiplimab. In embodiments,the antibody or active fragment thereof comprises CDRs that are at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe CDRs of Pembrolizumab. In embodiments, the antibody or activefragment thereof comprises CDRs that are at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDRs ofAtezolizumab. In embodiments, the antibody or active fragment thereofcomprises CDRs that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the CDRs of Avelumab. In embodiments, theantibody or active fragment thereof comprises CDRs that are at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe CDRs of Durvalumab. In embodiments, the antibody or active fragmentthereof comprises CDRs that are at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the CDRs of Pexidartinib. Inembodiments, the antibody or active fragment thereof comprises CDRs thatare at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the CDRs of PLX7486. In embodiments, the antibody or activefragment thereof comprises CDRs that are at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDRs of ARRY-382.In embodiments, the antibody or active fragment thereof comprises CDRsthat are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the CDRs of JNJ-40346527. In embodiments, the antibodyor active fragment thereof comprises CDRs that are at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CDRs ofBLZ945. In embodiments, the antibody or active fragment thereofcomprises CDRs that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the CDRs of Emactuzumab. In embodiments,the antibody or active fragment thereof comprises CDRs that are at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe CDRs of AMG820. In embodiments, the antibody or active fragmentthereof comprises CDRs that are at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the CDRs of IMC-CS4. Inembodiments, the antibody or active fragment thereof comprises CDRs thatare at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the CDRs of Cabiralizumab.

In some embodiments, the composition comprising an extracellular vesicleand an immunomodulating component is administered to a subject as acancer vaccine. In some of these embodiments, the composition isadministered to a subject as a personalized cancer vaccine. In someembodiments, the immunomodulating component is a tumor antigen or apeptide derived from a tumor antigen. Examples of suitable tumorantigens include: alpha-fetoprotein (AFP), carcinoembryonic antigen(CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16(MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor proteinp53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death ligand 1(PD-L1), programmed death ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72,HER2, GD2, cMET, EGFR, Mesothelin, VEGFR, alpha-folate receptor, CE7R,IL-3, Cancer-testis antigen, MART-1 gp100, and TNF-relatedapoptosis-inducing ligand. In certain embodiments, the tumor antigen isderived from a reference genome sequence. In certain embodiments, thetumor antigen is derived a genome sequence of the subject receiving thecomposition.

The cancers that can be treated with the composition include but are notlimited to the cancers listed in Table 5.

Methods of Modulating Gene Expression, Transcriptional Networks andPolarization in Macrophages

Some embodiments provide methods of modulating gene expression in amacrophage, comprising administering to a subject an extracellularvesicle comprising one or more immunomodulating component(s), whereinthe immunomodulating component is targeted to the gene, and wherein themodulation is equal to or greater than modulation produced byadministration of an equimolar amount of free immunomodulating componenttargeted to the gene. Also provided are methods of inhibiting geneexpression in a macrophage, comprising administering to a subject anextracellular vesicle comprising one or more immunomodulatingcomponent(s), wherein the immunomodulating component is targeted to thegene, and wherein the inhibition is equal to or greater than inhibitionproduced by administration of an equimolar amount of freeimmunomodulating component targeted to the gene. Some embodimentsprovide methods of repressing a downstream target of a gene in amacrophage, comprising administering to a subject an extracellularvesicle comprising one or more immunomodulating component(s), whereinthe immunomodulating component is targeted to the gene, and wherein therepression is equal to or greater than repression produced byadministration of an equimolar amount of free immunomodulating componenttargeted to the gene. Some embodiments provide methods of alteringpolarization of a population of macrophages, comprising administering toa subject an extracellular vesicle comprising one or moreimmunomodulating component(s), wherein the immunomodulating component istargeted to the gene, and wherein the alteration of polarization isequal to or greater than alteration of polarization produced byadministration of an equimolar amount of free immunomodulating componenttargeted to the gene. In some embodiments the extracellular vesicle isan exosome. In some embodiments, the immunomodulating component is anASO. In some embodiments the alteration in polarization is a change froman M2 to an M1 phenotype

Methods of Treating Fibrotic Conditions

Also, provided herein are methods of treating a fibrotic condition in asubject comprising administering to the subject in need thereof aneffective amount of a composition comprising exosomes comprising animmunomodulating component that repolarizes macrophages from the M2 toM1 phenotype. In some embodiments, the fibrotic condition is lungfibrosis, liver fibrosis, or pancreatic fibrosis. In certainembodiments, the liver fibrosis is non-alcoholic steatohepatitis, orNASH. See, e.g., See Mayo Clinic Staff. “Definition [of pulmonaryfibrosis]”. Mayo Foundation for Medical Education and Research. Archivedfrom the original on 15 Jul. 2014. Retrieved 26 Jul. 2014, availablefrom the world wide atwebmayoclinic.org/diseases-conditions/pulmonary-fibrosis/symptoms-causes/syc-20353690;Ferri F F. Idiopathic pulmonary fibrosis. In: Ferri's Clinical Advisor2016. Philadelphia, Pa.: Mosby Elsevier; 2016; Gross T J, Hunninghake GW (2001). “Idiopathic pulmonary fibrosis”. N Engl J Med. 345 (7):517-525; Friedman L S (2014). Current medical diagnosis and treatment2014. [S.1]: Mcgraw-Hill. pp. Chapter 16. Liver, Biliary Tract, &Pancreas Disorders; Chalasani N, Younossi Z, Lavine J E, Charlton M,Cusi K, Rinella M, Harrison S A, Brunt E M, Sanyal A J (January 2018).“The diagnosis and management of nonalcoholic fatty liver disease:Practice guidance from the American Association for the Study of LiverDiseases”. Hepatology. 67 (1): 328-357; Xue J, Sharma V, Hsieh M H,Chawla A, Murali R, Pandol S J, Habtezion A. Nat Commun. 2015 May 18;6:7158.

Modes of Administration

In some embodiments, the composition is administered intravenously tothe circulatory system of the subject. In some embodiments, thecomposition is infused in suitable liquid and administered into a veinof the subject.

In some embodiments, the composition is administered intra-arterialy tothe circulatory system of the subject. In some embodiments, thecomposition is infused in suitable liquid and administered into anartery of the subject.

In some embodiments, the composition is administered to the subject byintrathecal administration. In some embodiments, the composition isadministered via an injection into the spinal canal, or into thesubarachnoid space so that it reaches the cerebrospinal fluid (CSF).

In some embodiments, the composition is administered to the subject byintranasal administration. In some embodiments, the composition can beinsufflated through the nose in a form of either topical administrationor systemic administration. In certain embodiments, the composition isadministered as nasal spray.

In some embodiments, the composition is administered to the subject byintraperitoneal administration. In some embodiments, the composition isinfused in suitable liquid and injected into the peritoneum of thesubject. In some embodiments, said intraperitoneal administrationresults in distribution of the composition (e.g., the extracellularvesicles in the composition) to the lymphatics. In some embodiments,said intraperitoneal administration results in distribution of thecomposition (e.g., the extracellular vesicles in the composition) to thethymus, spleen, and/or bone marrow. In some embodiments, saidintraperitoneal administration results in distribution of thecomposition (e.g., the extracellular vesicles in the composition) to oneor more lymph nodes. In some embodiments, said intraperitonealadministration results in distribution of the composition (e.g., theextracellular vesicles in the composition) to one or more of thecervical lymph node, the inguinal lymph node, the mediastinal lymphnode, or the sternal lymph node. In some embodiments, saidintraperitoneal administration results in distribution of thecomposition (e.g., the extracellular vesicles in the composition) to thepancreas.

In some embodiments, the composition is administered to the subject byperiocular administration. In some embodiments, the composition isinjected into the periocular tissues. Periocular drug administrationincludes the routes of subconjunctival, anterior sub-Tenon's, posteriorsub-Tenon's, and retrobulbar administration.

In some embodiments, the composition is administered into the samesubject by multiple routes of administration. In some embodiments, saidmultiple routes of administration comprise intravenous administration,intra-arterial administration, intrathecal administration, intranasaladministration, intraperitoneal administration, and/or periocularadministration. In a preferred embodiment, said multiple routes ofadministration comprise intravenous administration and intraperitonealadministration.

In certain embodiments, the dosage of the extracellular vesicles isbetween 1 ng to 10 ng, 10 ng to 100 ng, 100 ng to 1 μg, 1 μg to 5 μg, 5μg to 10 μg, 10 μg to 50 μg, 50 μg to 75 μg, 75 μg to 100 μg, 100 μg to150 μg, 150 μg to 200 μg, 200 μg to 300 μg, 300 μg to 500 μg, 500 μg to1 mg, or 1 mg to 10 mg.

The compositions can be administered once to the subject. Alternatively,multiple administrations can be performed over a period of time. Forexample, two, three, four, five, or more administrations can be given tothe subject. In some embodiments, administrations can be given asneeded, e.g., for as long as symptoms associated with the disease,disorder or condition persists. In some embodiments, repeatedadministrations can be indicated for the remainder of the subject'slife. Treatment periods can vary and can be, e.g., no longer than ayear, six months, three months, two months, one month, two weeks, oneweek, three days, two days, or no longer than one day.

In certain embodiments, doses of extracellular vesicles are administeredat intervals such as once daily, every other day, once weekly, twiceweekly, once monthly or twice monthly.

In some embodiments, the pharmaceutical composition is administered at afrequency sufficient to effectively increase the concentration of theimmunomodulating component in the target cell or tissue above a levelthat is associated with a symptom of the disease, disorder or condition.

In some embodiments, the compositions are administered at least twiceover a treatment period such that the disease, disorder or condition istreated, or a symptom thereof is ameliorated. In some embodiments, thecompositions are administered at least twice over a treatment periodsuch that the disease, disorder or condition is treated or a symptomthereof is prevented. In some embodiments, the pharmaceuticalcomposition is administered a sufficient number of times over atreatment period such that a sufficient amount of immunomodulatingcomponent is delivered to the target cell or tissue during the treatmentperiod. In some embodiments, the pharmaceutical composition isadministered a sufficient number of times over a treatment period suchthat a sufficient amount of immunomodulating component is delivered tothe target cell or tissue during the treatment period such that one ormore symptoms of the disease, disorder or condition is prevented,decreased, ameliorated or delayed. In some embodiments, increasing theimmunomodulating component concentration in the target cell or tissueincludes increasing the peak concentration, while in others it includesincreasing the average concentration. In some embodiments, a substantialincrease during the treatment period can be determined by comparing apretreatment or post-treatment period in the subject, or by comparingmeasurements made in a population undergoing treatment with a matched,untreated control population.

In some embodiments, the pharmaceutical composition is administered asufficient number of times per treatment period such that theconcentration of immunomodulating component in the target cell or tissueis increased for at least about one week, two weeks, three weeks, fourweeks, one month, two months, three months, four months, five months,six months or greater than six months. In some embodiments, thepharmaceutical composition is administered a sufficient number of timesper treatment period such that the concentration of immunomodulatingcomponent in the target cell or tissue is increased for a period of timeat least as long as the treatment period.

In some embodiments, the time interval between repeated administrationswithin a treatment period is no longer than the period in which thenumber of extracellular vesicles in circulation is reduced to less thanabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% of the number of extracellular vesiclespresent in the administered pharmaceutical composition.

In some embodiments, the methods of treatment further comprise one ormultiple doses of non-therapeutic extracellular vesicles prior to theinjection of a suitable therapeutic dose of extracellular vesiclesharboring a therapeutic agent. In certain embodiments, thenon-therapeutic extracellular vesicle is administered separately to andat a different dosage than the therapeutic extracellular vesicles. Incertain embodiments, the dosage of the non-therapeutic extracellularvesicle is greater than the dosage of the therapeutic extracellularvesicle. In certain other embodiments, the dosage of the non-therapeuticextracellular vesicle is smaller than the dosage of the therapeuticextracellular vesicle. In certain embodiments, the dosage of thenon-therapeutic extracellular vesicle is the same as the therapeuticextracellular vesicle. In various embodiments, the methods ofnon-therapeutic extracellular vesicles prior to injection of a suitabledose of therapeutic extracellular vesicles reduce the update of thetherapeutic extracellular vesicles in the liver, lung, and/or spleen.See co-owned PCT application PCT/US2017/047794, incorporated herein byreference for all purposes.

An effective amount of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the extracellular vesicle (e.g., size, andin some cases the extent of molecules to be delivered) and otherdeterminants. In general, an effective amount of the compositionprovides efficient cellular response of the target cell. Increasedefficiency can be demonstrated by increased cell transfection (i.e., thepercentage of cells transfected with the extracellular vesicleconstituents), increased cellular response or reduced innate immuneresponse of the host subject.

The dosing and frequency of the administration of the extracellularvesicles and pharmaceutical compositions thereof can be determined,e.g., by the attending physician based on various factors such as theseverity of disease, the patient's age, sex and diet, the severity ofany inflammation, time of administration and other clinical factors. Inan example, an intravenous administration is initiated at a dose whichis minimally effective, and the dose is increased over a pre-selectedtime course until a positive effect is observed. Subsequently,incremental increases in dosage are made limiting to levels that producea corresponding increase in effect while taking into account any adverseeffects that can appear.

Additional Embodiments

Other aspects and embodiments are provided in the following numbereditems.

1. An extracellular vesicle comprising one or more nucleic acidmolecules that inhibits at least one gene and thereby increasesmacrophage polarization from the M2 to M1 phenotype.2. The extracellular vesicle of embodiment 1, wherein the extracellularvesicle is an exosome.3. The extracellular vesicle of embodiment 1 or 2, wherein the nucleicacid is an inhibitory RNA.4. The extracellular vesicle of any one of embodiments 1-3, wherein theinhibitory RNA is an antisense RNA, siRNA, ShRNA, miRNA, an lncRNA orpre-miRNA.5. The extracellular vesicle of any one of embodiments 1-4, wherein theat least one gene is selected from the group consisting of.

KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3,STAT6, n-MYC, and c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, Glutaminase,and PKM2.

6. The extracellular vesicle of embodiment 5, wherein the gene is KRAS.7. The extracellular vesicle of embodiment 6, wherein the nucleic acidis an inhibitory RNA that targets wild-type human KRAS.8. The extracellular vesicle of embodiment 7, wherein the inhibitory RNAalso targets mouse Kras^(G12D).9. The extracellular vesicle of any one of embodiments 1-8, wherein themacrophage is a tumor resident macrophage.10. The extracellular vesicle of embodiment 9, wherein the tumor is apancreatic tumor.11. The extracellular vesicle of any one of embodiments 1-10, furthercomprising an additional immunomodulating component.12. The extracellular vesicle of embodiment 11, wherein the additionalimmunomodulating component is a small molecule drug, an antibody or atherapeutic protein.13. The extracellular vesicle of embodiment 12, wherein the antibody isan immune checkpoint inhibitor.14. A pharmaceutical composition comprising the extracellular vesicle ofany one of embodiments 1-13.15. A method of treating a disease in a patient in need thereofcomprising administering the extracellular vesicle of any one ofembodiments 1-13 or the pharmaceutical composition of embodiment 14 tothe patient, thereby treating the disease in the patient.16. The method of embodiment 15, wherein the disease is a cancer.17. The method of embodiment 15 or 16, wherein the patient is human.18. The method of any one of embodiments 1-17, wherein the nucleic acidis an inhibitory RNA targeting a proto-oncogene.19. The method of embodiment 18, wherein the proto-oncogene is humanKRAS.20. The method of embodiment 19, wherein the cancer is pancreaticcancer.21. The method of any one of embodiments 15-20, further comprisingperforming at least a second therapy.22. The method of embodiment 21, wherein the second therapy comprises asurgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonaltherapy, or immunotherapy.23. The extracellular vesicle of any one of the above embodimentswherein the M2 macrophage is a tumor associated macrophage selected fromthe group consisting of a M2a, M2b, and M2c macrophage.24. The extracellular vesicle of any one of the above embodimentswherein the M1 macrophage exhibits increased secretion of inflammatorycytokines and chemokines selected from the group consisting of INFγ,IL-12, IL-23, TNFα, IL-6, IL-1, CSCL9, CXCL10 and CXCL11 compared to theM2 macrophage prior to polarization.25. The extracellular vesicle of any one of the above embodimentswherein the M1 macrophage exhibits decreased secretion ofimmunosuppressive cytokines and chemokines selected from the groupconsisting IL-10, TGFβ, PGE2, CCL2, CCL17, CCL18, CCL22 and CCL24compared to the M2 macrophage prior to polarization.26. The extracellular vesicle of any one of the above embodimentswherein the M1 macrophage expresses increased tumor associated antigencompared to the M2 macrophage prior to polarization.27. The extracellular vesicle of any one of the above embodimentswherein the M1 macrophage increases stimulation of CD8⁺ T-Cells and/orNatural Killer cells compared to the M2 macrophage prior topolarization.28. A method of treating pancreatic cancer in a subject comprising:administering to the subject an extracellular vesicle comprising aninhibitory RNA targeting human wild-type KRAS; wherein the treatmentincreases the percentage of polarization of tumor-resident macrophagesfrom the M2 to M1 phenotype to a greater level than that observed in apatient treated with an inhibitory RNA targeting human KRAS^(G12D).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations can be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt,nucleotide(s); and the like.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 21th Edition (Easton, Pa.: Mack PublishingCompany, 2005); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1: Knockdown of Transcription Factors in M2 Macrophages Methods:

Monocyte Isolation from PBMC:

-   -   50 ml of Buffy coat was received    -   15 ml of ficol was pipetted into 50 ml STEMCELL SepMate tubes    -   Buffy coat was diluted in PBS 2 mM EDTA, such that final volume        was 250 mL    -   The diluted buffy coat sample was poured into Sepmate tubes        until final volume in the tube was ˜45-50 ml    -   The tubes were centrifuged at 1000 g for 15 min with brake on 1    -   Plasma was aspirated from the spun tubes, and the buffy coat        layer was collected with a pipetaid into a 50 ml conical tube.        Tubes were filled with PBS/EDTA and centrifuged to pellet the        cells at 500 g for 3 min.    -   If red blood cells were highly present, the cells were        resuspended in 10 ml of ACK lysing buffer (ThermoFisher, Catalog        no. A1049201) and incubated at room temperature for 3 minutes        (manufacturers protocol). The tubes were filled with PBS/EDTA        and centrifuge at 500 g for 3 minutes.    -   Tubes were spun down 5 min at 400×g, and pellet was washed with        PBS EDTA once more and re-pelleted    -   The pellet was resuspended in RoboSep buffer (STEMCELL, catalog        no. 20104) and cells were counted using a 1:1 dilution with        trypan blue (Thermofisher) (˜500 million PBMCs yielded from one        buffy coat)    -   CD14+ monocytes were isolated using EasySep Human Monocyte        Enrichment Kit (STEMCELL, Catalog no. 19059RF), according to        manufacturer's protocol (10% of total cells were isolated as        monocytes)    -   5 million monocytes were plated in one plate in RPMI 10% FBS 1%        Anti Anti+40 ng/ml M-CSF        Macrophage differentiation and polarization: (Adaptedfrom        https://www.atsjoumals.org/doi/full/10.1165/rcmb.2015-0012OC)    -   The plated cells were cultured in RPMI-1640 10% FBS 1% Anti Anti        1% PenStrep+40 ng/ml M-CSF (Biolegend) for 5-6 days at 37 C, 5%        CO2. 5 ml of the same fresh media was added on Day 1 and 3 post        plating. At day 5, polarizing cytokines were added as follows        (with 40 ng/ml MSCF; all cytokines added at 20 ng/ml):    -   M0: no cytokines    -   M2a: IL-4    -   M2c: IL-10    -   M2++:114, 1110, TGFb    -   ML: IFN-g+LPS (100 ng/ml)    -   TAM: 75% Panc-1 supernantant    -   Cells were incubated with cytokines for 24 hours    -   At Day 6, media was aspirated, cells were wash with PBS and then        10 ml of PBS 5 mM EDTA (ice cold) was added per plate, and        incubate at 4 C for 30 min    -   Cells were gently scraped off plates, and counted.    -   50,000 cells per well were then plated in a 96 well plate in in        RPMI-1640 10% FBS 1% Anti Anti 1% PenStrep+40 ng/ml M-CSF        (Biolegend) with the respective cytokines for different        macrophage populations (see above) for 24 hours, after which        treatments were initiated.

Exosome Purification:

Exosomes were collected from the supernatant of high density suspensioncultures of HEK293 SF cells after 9 days. The supernatant was filteredand fractionated by anion exchange chromatography and eluted in a stepgradient of sodium chloride. The peak fraction with the highest proteinconcentration contained exosomes and contaminating cellular components.The peak fraction was isolated and further fractionated on an Optiprep™(60% iodixanol w/v) density gradient by ultracentrifugation.

The exosome fraction was concentrated by ultracentrifugation in a 38.5mL Ultra-Clear (344058) tube for a SW 32 Ti rotor at 133, 900×g for 3hours at 4° C. The pelleted material was resuspended in 1 mL PBS and 3mL of Optiprep™, bringing the final iodixanol concentration to 45%. Forthe Optiprep™ gradient, a 4-tier sterile gradient was prepared with 4 mLof 45% iodixanol containing the resuspended material, 3 mL 30%iodixanol, 2 mL 22.5% iodixanol, 2 mL 17.5% iodixanol, and 1 mL PBS in a12 mL Ultra-Clear (344059) tube for a SW 41 Ti rotor. The Optiprep™gradient was ultracentrifuged at 150,000×g for 16 hours at 4° C. toseparate the exosome fraction. Ultracentrifugation resulted in a TopFraction known to contain exosomes, a Middle Fraction containing celldebris of moderate density, and a Bottom Fraction containing highdensity aggregates and cellular debris. The exosome layer was thengently collected from the top ˜3 mL of the tube.

The exosome fraction was diluted in ˜32 mL PBS in a 38.5 mL Ultra-Clear(344058) tube and ultracentrifuged at 133, 900×g for 3 hours at 4° C. topellet the purified exosomes. The pelleted exosomes were thenresuspended in a minimal volume of PBS (˜200 μL) and stored at 4° C.

Many solid tumors are characterized by a myeloid-rich cellularinfiltrate, often comprising tumor-associated macrophages, characterizedas having an alternatively-activated, or M2, phenotype. M2 macrophagesexpress high levels of phosphorylated STAT3 and STAT6, which promote theexpression of the metabolic enzyme Arginase (Arg1). To demonstrate thatknockdown of critical M2 genes alters the M2 phenotype of in vitrodifferentiated macrophages, murine RAW264.7 macrophages were polarizedto an M2 state as described above. Cultured polarized macrophages weretransfected with increasing amounts of siRNA targeting STAT3, STAT6,CEBPβ-1, CEBPβ-2, Pi3Kγ, or KRAS (12.5 nM, 25 nM, 50 nM, and 100 nM).Each of the genes was repressed in a dose-dependent manner (FIG. 1), andthe knockdown of each M2 gene led to the concomitant reduction of Arg1(FIG. 2), demonstrating that the M2 phenotype could be altered byreducing the expression of upstream regulators.

Example 2: Differential Exosome Uptake by Macrophage Subtypes

Macrophage subtypes are characterized by alterations in metabolic andother phenotypic activity. To understand whether certain macrophagesubtypes preferentially take up exosomes, six macrophage subpopulations(M0, M1, M2a, M2c, M2++, and TAM) were incubated with increasing levelsof HEK293SF-derived exosomes engineered to express lumenal GFP. Exosomeuptake was determined by measuring total cellular GFP intensity over 36hours in an IncuCyte® live cell analysis system (Essen Bioscience). Asshown in FIG. 3, while all macrophage subtypes showed uptake ofexosomes, M0 macrophages were the least efficient at taking up exosomes,while M2++ macrophages were substantially more efficient at taking upexosomes compared to other macrophage subtypes.

Since M2 macrophages are efficient at taking up exosomes, weinvestigated whether ASO-loaded exosomes would be more efficient attargeting intracellular macrophage targets (e.g., more efficient atknocking down expression of the target gene, and down-stream effectormolecules within the gene's signaling pathway) than ASOs alone.2′-methoxyethyl (MOE) single stranded DNA/RNA ASOs targeting STAT3 andcarrying a Cy5 fluorescent tracer and 5′ cholesterol linker weregenerated. HEK293SF exosomes were mixed with the cholesterol-taggedfluorescent ASOs, and free ASOs were removed by ultracentrifugation andremoval of the ASO-containing supernatant. M2 and M0 macrophages wereplated at equal density and incubated with matched concentrations offree ASOs or ASO-loaded exosomes (Exo-ASO) as measured by totalfluorescence intensity. Total cellular fluorescence was measured over 48hours and quantified using an IncuCyte® live-cell analysis system. Asshown in FIG. 4, M2 macrophages more readily took up both free ASOs andExo-ASO compared to M0 macrophages. Interestingly, Exo-ASOs were takenup more efficiently by both M0 and M2 macrophages, suggesting thatdelivery of ASOs to M2 macrophages may be enhanced by loading onexosomes.

To determine whether exosomes or Exo-ASO were differentially taken up bymacrophages as compared to other cell types in vivo, naïve mice weredosed intraperitoneally with either 1×10¹¹ or 1×10¹² GFP-containingexosomes. One hour post-injection, total peritoneal cells were isolatedand characterized by flow cytometry for GFP positivity among differentimmune cell subsets, i.e., B cells, Dendritic cells, neutrophils,Natural Killer (NK) cells, T cells and macrophages. As shown in FIG. 5A,at both doses, macrophages were the predominant cell type to take upfluorescent exosomes. To determine whether this difference occurred in adisease setting, B16F10 tumor-bearing mice were injected with a singledose of Cy5-labeled Exo-ASO or native unlabeled exosomes. The injectedtumors were isolated, dissociated, and measured by flow cytometry. Asshown in FIG. 5B, macrophages took up Exo-ASO much more readily thantumor cells (˜100-fold greater overall Cy5 signal), suggesting thatdelivery of Exo-ASO can reach macrophage populations in several complexcellular mixtures. Furthermore, the preferential uptake of Exo-ASO bymacrophages as compared to the other tested cell types (B cells,Dendritic cells, neutrophils, Natural Killer (NK) cells and T cells)enables methods of ASO delivery targeted to macrophage cells, comprisingadministering an Exo-ASO composition to a subject, wherein theadministered composition is preferentially taken up by macrophagespresent in the subject (as compared to uptake by other immune cellsubsets, e.g., B cells, Dendritic cells, neutrophils, Natural Killer(NK) cells and T cells). This enhanced macrophage specificity of theExo-ASO composition improves safety of the administered composition byreducing effects in off-target cells.

Example 3: Exosome-Mediated Delivery of Antisense Oligos PotentlyDysregulates Macrophage Transcriptional Networks and Alters Polarization

The previous experiments suggest that exosome-mediated delivery of ASOsmay be an effective method of altering macrophage gene expressionpatterns. HEK293SF-derived exosomes purified according to the abovemethods were loaded with one of five different cholesterol-tagged ASOstargeting STAT3 (4.1, double-stranded MOE chemistry; 5.1 and 5.2,single-stranded O-methyl chemistry; 5.3 and 5.4, single-stranded MOEchemistry). The ASO sequences are shown in Table 0. M2 polarized murineRAW264.7 cells were incubated with 5 μM free cholesterol-tagged ASO oreither 1×10⁵ or 1×10⁶ exosomes loaded with the cholesterol-tagged ASO.STAT3 transcript levels were measured 24 hours after treatment,revealing comparable or superior knockdown of STAT3 with Exo-ASOtreatment as compared to free ASO. Unmodified exosomes incubated withthe polarized macrophages had no impact on STAT3 expression (FIG. 6). Toexamine downstream targets of STAT3, ARG1 transcript levels weremeasured 48 hours after treatment. As shown in FIG. 7, STAT3 knockdownwith free ASO or Exo-ASO led to robust repression of Arg1, in severalcases by more than 90% (FIG. 7). M2-polarized RAW264.7 cells wereincubated with high (4 μM) and low (0.4 μM) ASO or Exo-ASO for STAT3,KRAS, and C/EBPβ. As shown in FIGS. 8A-C, Exo-ASO samples were equally(STAT3) or more potent (KRAS, C/EBPβ) at all tested doses (FIGS. 8A-C).The ASO sequences are shown in Table 0.

This result demonstrates that Exo-ASO is a potent modulator ofmacrophage expression networks, and may provide a superior,differentiated modality, as compared to administration of free ASO forvarious therapeutic applications. One such method to modulate geneexpression in a macrophage, comprising administering to a subject anExo-ASO, wherein said ASO is targeted to the gene, and wherein themodulation is equal to or greater than modulation produced byadministration of an equimolar amount of free ASO targeted to the gene.Another embodiment comprises a method of inhibiting gene expression in amacrophage, comprising administering to a subject an Exo-ASO, whereinsaid ASO is targeted to the gene, and wherein the inhibition is equal toor greater than inhibition produced by administration of an equimolaramount of free ASO targeted to the gene. Another embodiment comprises amethod of repressing a downstream target of a gene in a macrophage,comprising administering to a subject an Exo-ASO, wherein said ASO istargeted to the gene, and wherein the repression is equal to or greaterthan repression produced by administration of an equimolar amount offree ASO targeted to the gene. Yet another embodiment comprises a methodof altering polarization of a population of macrophages, comprisingadministering to a subject an Exo-ASO, wherein said ASO is targeted to agene expressed in the macrophages, and wherein the alteration ofpolarization is equal to or greater than alteration of polarizationproduced by administration of an equimolar amount of free ASO targetedto the gene. In such embodiments the alteration in polarization can be achange from an M2 to an M1 phenotype.

To test the role of Exo-ASO in human cells, primary human macrophageswere polarized to an M2 phenotype and treated with varying doses of afree STAT3 ASO or STAT3 Exo-ASO. Using macrophages from three separatehuman donors, Exo-ASO was consistently more potent in repressing STAT3transcript levels 24 hours after treatment (FIG. 9A). The downstreamhuman marker of M2 polarization, CD163, was also more dramaticallymodulated by Exo-ASO compared to free ASO (FIG. 9B). Human M2macrophages were also treated with 4 μM free ASO or Exo-ASO for HIF1α,Pi3Kγ, CEBP/β, STAT6, and STAT3. As shown in FIG. 10, all treatmentgroups resulted in the repression of the target gene, but in all casesExo-ASO was more potent than the free ASO treatment. Importantly,Exo-ASO knockdown of any of the macrophage targets led to robustreduction in CD163 expression (FIG. 11), demonstrating thatexosome-mediated delivery of ASOs potently and reproducibly disruptsimportant macrophage signaling networks.

A functional consequence of M2 macrophage polarization is a reduction inpro-inflammatory cytokines, which contribute to a pro-tumorigenicmicroenvironment. Conversion of macrophages from M2 to M1 phenotypeshould therefore result in enhanced pro-inflammatory signaling.Conversion of M2 macrophages to the M1 state can be induced by LPS,which leads to the induction of cytokines including IL-12 and IL-23.STAT3 is a negative regulator of this cytokine expression network, andthus we tested whether STAT3 inhibition in the presence of LPS canfurther enhance the production of pro-inflammatory cytokines.M2-polarized human macrophages were treated with 4 μM free STAT3 ASO, 4μM STAT3 Exo-ASO, 4 μM scrambled Exo-ASO, native exosomes, or C188-9, asmall molecule inhibitor of STAT3. 24 hours later, media was exchangedto media containing 10 ng/ml LPS, and supernatant was isolated 24 hourslater. As shown in FIG. 12, LPS induced the secretion of IL-12 and IL-23(LPS 10 ng/ml group vs. No LPS group). Treatment with STAT3 Exo-ASO butnot free STAT3 ASO further enhanced the expression of each cytokine,suggesting that Exo-ASO can more potently lead to the promotion of an M1phenotype in response to relevant immuno-modulatory signals.

Dysregulation of STAT3, STAT6, and the other macrophage modulatorypathways leads to broad changes in gene expression patterns. Tounderstand the differentiated global impact of Exo-ASO treatment,NanoString mRNA analysis was carried out on human M2 macrophages attheir steady state and in response to treatment with unmodified exosomes(EV only), and concentration-matched ASO and Exo-ASO. Normalized mRNAcounts using the nCounter® Human Myeloid Innate Immunity Paneldemonstrated that STAT3 transcripts were reduced after both free ASO andExo-ASO treatment (FIG. 13A). Downstream markers CD163 (FIG. 13D), TGFβ(FIG. 13C), and STAT6 (FIG. 13D) were all potently downregulated afterASO treatment.

Robust repression of TGFβ(<85%) is a critical regulator of M2 macrophagepolarization, (Yang and Zhang Journal of Hematology & Oncology (2017)10:58). We examined the impact of STAT3, STAT6, and CEBP/β Exo-ASOtreatment on TGFβ expression. Human M2-polarized macrophages weretreated with exosomes loaded with ASOs against STAT3 (both MOE and LNAchemistry), STAT6 (MOE chemistry), and CEBP/β (MOE chemistry). In allcases, TGFβ levels were dramatically reduced after treatment withsub-micromolar levels of ASO (FIG. 14). These results demonstrate thatexosome-mediated delivery of ASOs against numerous macrophagepolarization regulators can lead to robust, stable dysregulation ofcellular programs that may be impactful in the treatment of numerouscancers. Importantly, each of the macrophage targets, including thenovel recognition of KRAS as a regulator of this process, have beencharacterized as “undruggable” targets, in that their role in diseaseand other cellular processes is well-recognized, but safe, effectivetreatment has eluded classical approaches of drug development.

Example 4: Inhibition of Wild-Type KRAS Leads to Increased MacrophagePolarization and Reduced Tumor Size In Vivo in an Orthotopic Mouse Modelof Pancreatic Cancer Methods:

Production and Isolation of KRAS Exosomes:

Electroporation methods are used to insert KRAS siRNA constructs intoexosomes without functionally damaging exosomes. Exosomes are isolatedfrom human embryonic kidney (HEK) 293SF suspension cells grown inchemically-defined medium using established ultracentrifugation methods(Kahlert et al., 2014). The purity and homogeneity (80-150 nm diameterparticles) of the exosomes is validated by Nanosight™ measurements,transmission electron microscopy, and CD9 immunogold labeling. Sucrosegradient ultracentrifugation and qPCR are also performed to validate thepresence and abundance of the KRAS siRNA. Scrambled siRNA containingexosomes are also generated.

RNAi Strategies.

The KRAS siRNA sequence targeted against mouse wild-type KRAS comprisesa TT nucleotide overhang to promote silencing efficiency. The RNAi isalso labeled with an Alexa Fluor® 647 fluorophore at the 3′ end on thesense strand to track its delivery.

Mice and Imaging.

Female athymic nu/nu mice (Charles Rivers) between 4-6 weeks of age arehoused in individually ventilated cages on a 12 h light-dark cycle at21-23° C. and 40%-60% humidity. Mice are allowed free access to anirradiated diet and sterilized water. Under general anesthesia,tumorigenic human pancreas Panc-1 (Kras^(asp12) (Rejiba et al., 2007;Sun et al., 2001)) cells or BXPC-3 cells (10⁶, resuspended in 10 μl PBS)are injected into the tail of the pancreas using a 27-gauge syringe. Fororthotopic tumor size/volume analyses, Living Image version 4.4 (CaliperLife Sciences) is used to quantify all tumor calculations. A circularregion of interest (ROI) around the pancreas and tumor is defined andset as a standard to compare all the images within the same experimentalgroup. In addition, exposure conditions (time, aperture, stage position,binning) are kept identical for all measurements in all experimentalgroups. Subsequent tumor measurements (p/sec/cm²/sr) are then obtainedunder the same conditions for all experimental groups. The mice areimaged regularly and randomly divided into groups for treatments. Micereceive 2×10⁸ exosomes i.p. in 100 μl volume of PBS every other day.Exosomes are electroporated with 2 μg of siRNA and washed with PBS priorto injection.

Histology, Histopathology, and Immunohistochemistry.

Tissues are fixed in formalin and processed for paraffin embedding.Tissue sections of 5 μm thickness are cut and stained for hematoxylinand eosin (H&E) and Masson's trichrome (MTS) (Leica). Forhistopathological scoring, H&E stained slides were scored based on themorphological stages of pancreas cancer: Normal, pancreaticintraepithelial neoplasia (PaNIN) and pancreatic ductal adenocarcinoma(PDAC). For each tissue section, a percentage score for each of thethree stages (Normal, PaNIN, PDAC) is obtained manually in a blindedfashion by experts in pancreas histology, which was then averaged togive an overall score out of 100 for each cohort. An average of thesepercentage scores is then taken for each mouse in the respectivecohorts. Tumor tissue sections are also stained for macrophages (witheither pan-macrophage markers (e.g., F4/80 anibody and/or M2 and M1specific cell markers for detection of tumor resident macrophages).

Assays for Macrophage Phenotype Characterization.

Blood samples and tumor samples are collected from mice. Macrophages areisolated and stained for analyses of M1 and M2 markers by flowcytometry. Macrophages are stained for pan-macrophage markers as well asM2 phenotype cell surface markers (e.g., YM1, FIZZ1, Dectin-1 and/orMGL) and M1 phenotype cell surface markers. M2 and M1 macrophages arethen counted and sorted for further analyses, such as quantitative PCRto detect cytokine and/or miRNA expression of miRNAs associated witheither M1 and/or M2 phenotype).

The results from the xenograft experiments and orthotopic tumor volumeanalyses confirm that exosomes comprising KRAS wild-type siRNAeffectively reduce the size of pancreatic tumors and increase the numberof M1 phenotype tumor associated macrophages compared to exosomesharboring a scrambled siRNA, indicating that administration of exosomescomprising inhibitory RNA targeted against human wild-type KRAS iseffective for the treatment of cancer.

Tables

TABLE 0 Sequences of antisense oligonucleotides (ASOs) Target SequenceSEQ ID NO: Stat3 TAAGCTGATAATTCAACTCA SEQ ID NO:1 Stat6TGAGCGAATGGACAGGTCTT SEQ ID NO:2 CebpB TGGATTTAAAGGCAGGCGGC SEQ ID NO:3Pi3Kγ TTGGGTAAAGTCGTGCAGCA SEQ ID NO:4 HIF1-α GTGCAGTATTGTAGCCAGGCSEQ ID NO:5 Kras GTAGCATGTAAATATAGCCC SEQ ID NO:6 All antisenseoligonucleotides have a phosphorothioate bond between each nucleotide

TABLE 1 Exosome lipids Lysobisphosphatidic acid Ganglioside GM3 24:1Sphingomyelin (SM) Ganglioside GM3 16:0 Ganglioside GM3 PE40:5Phosphatidylserine (PS) PE40:6 Phosphatidylinositol (PI) PE38:3Phosphatidylcholine (PC) PE38:4 Phosphatidylethanolamine (PE) PE36:1Lysophosphatidylcholine (LPC) PE36:2 Cholesterol (Chol) PE34:1Diacylglycerol (DG) PE34:2 PI18:0/20:3 PE-ether38:5 PI18:0/20:4PE-ether38:6 PI18:0/18:1 PE-ether34:1 PI18:1/18:1 PE-ether34:2PI18:0/16:0 PC34:1 PA18:0/18:1 PC36:4 PS18:0/18:1 PC34:3 BMP18:0/18:1PC32:0 BMP18:1/18:1 PC30:0 BMP18:1/16:0 SM24:1 CL(18:1)3/16:1 SM16:0CL(18:1)2/(16:1)2 Dihydrosphingomyelin16:0

TABLE 2 Exosome polypeptides ACLY TCP1 ACTR1A LY75 ACTB PRDX2 THOC4ABCC1 ACTG1 TSPAN6 INADL MYO1E ALB CCT3 CTDSPL NACA ALDOA TSTA3 ZMPSTE24NAP1L4 ALDOB TUBA3C DNAJA2 NCL AKR1B1 HIST1H2AK NDRG1 NEDD8 AMBPHIST1H2AJ RAP GEF3 YBX1 ANPEP HIST1H2AB SP ON2 PA2G4 ANXA2 HI ST2H2ACUBAC1 PECAM1 ANXA3 IFITM1 N4BP2L2 PFAS ANXA4 PDXK CAP1 SERPINB9 ANXA5LIN7A VAT1 PI4KA ANXA6 BUB3 NEBL PLAT ANXA7 MAP4K4 DCTN2 PLCG2 ANXA11EDIL3 ARPC1A PPA1 ATP6AP2 C6orf108 PPP2CA CAPZB PSME3 SMC2 PRKCB CD63TUBB3 AHSA1 PSMA6 CD81 IFITM3 STAMBP PSMA7 CKB ACAA2 PMVK PSMB8 CLU CCT7GIPC1 PSMB9 CLIC1 CCT4 HBS1L PSMD7 TPP1 IFITM2 NCKAP1 P SME1 CLTC GNA13ALDH1L1 PTPRA CNP RUVBL2 FTCD RAC2 COL6A1 PRSS23 FGL2 RPL3 CR1 ACOT7CFHR3 RPL4 CTNND1 CCT5 MMP24 RPL5 ACE DIP2C COPS8 RPL11 DDT ASCC3L1CKAP4 RPL22 DEFA1 TNIK ClOorf116 RPL24 DEFA3 NEDD4L SLC27A2 RPL27 DNAH8NCSTN MID2 RPL30 DPEP1 TSPAN15 KIF3A RPL28 DPP4 PLXNB2 NUDT5 RPL31EEF1A1 SDCBP2 TREH RPL34 EEF2 IGKV1-5 CEP250 RPL35A EGF IGHV4-31 PDCD10RPL37A EIF5A IGKV3-20 PADI2 RPS2 ENO1 IGKV2-24 PACSIN2 RPS3A ENO3 MINK1CHP RPS5 ENPEP IGKα SNF8 RP S9 STOM VPS36 DDX19B RPS19 EPS8 DERA SCN11ARPS25 FABP3 GOLGA7 LYPLA2 RPS26 FGA KRT76 PARK7 RPS28 MLANA EIF3EIPCOBLL1 RPS29 FN1 LSR CNKSR2 RSU1 FTL TUBA8 ENPP4 SARS FUS RAB4B RAB3GAP1SLAMF1 GAA SETD4 AKR7A3 SLC1A4 GAPDH TOLLIP SPEN SLC2A3 GDI2 PLEKHB2GANAB SNRPD2 GGT1 VPS37C MGRN1 SPINK1 GLB1 LIN7C CUX2 SPN GLG1 H2AFJDNAJC13 STK10 GNA11 CAND1 ZCCHC11 STXBP3 GNAI1 PLSCR3 PHF15 TALD01 GNAI2KIAA1199 KIAA0841 TNFAIP3 GNAI3 GNB4 ARHGEF12 TPM3 GNAS MYH14 COTL1 TPM4GNB1 TSPAN14 ANGPTL2 TYK2 GNB2 NCALD DDAH2 VIM GNG7 REG4 HEBP2 WARS SFNVPS25 CD2AP WAS GPI TUBB6 PLD3 LAT2 GSTA1 TUBA1C TMEM2 HI5T1H2BL GSTA2TNKS1BP1 SH3BP4 STX7 GSTA3 FAM125B BHMT2 CPNE1 GSTM3 LRSAM1 GCA RPL14GSTP1 HIST3H2A MXRA5 PDCD5 GUSB TUBA3E AHCTF1 SYNGR2 HIST1H2AD TUBA3DPTPN23 RPL23 HLA-A DCD DAK RAB9A HLA-B HIS T4H4 ACOT11 IGSF2 HLA-DQB1ALDH16A1 APPL1 EEF1E1 HLA-DRA RP S4Y2 PHGDH SCAMP2 HLA-DRB1 MYL6B TIAM2SCAMP3 HLA-DRB5 BRI3BP KCNG2 DPP3 HPGD AGR3 CYFIP2 ARPC1B HRAS EEF1AL3GHITM PDIA6 HSPA1A KRT28 C11orf54 WASF2 HSPA1B KRT24 DBNL ANP32B HSPA8RPLP0-like ATAD2 PAICS HSP90AA1 RPSAP15 PHPT1 AHCYL1 RANP1 C16orf80VAMP5 KRT1 PCSK9 OLA1 41891 KRT9 METRNL ZDHHC1 HSPH1 KRT10 LOC284889SNX12 SUB1 LDHA KRT6C PSAT1 CDC37 LDHB KRT79 NT5C CORO1A TACSTD1 RAB43EHD2 CD300A MCAM KRT27 TAX1BP3 TMC6 MDH1 ACTBL2 CRNN RFTN1 MEP1ARP11-631M21.2 NOX3 SCRIB MSN TUBB2B ATP6V0A4 SERBP1 2-Sep KRT77 ITSN2TTLL3 PGAM1 AGRN GEMIN4 CACYBP PGK1 RAB15 LAP3 SIT1 PKM2 LOC388524 CRYL1SLC43A3 PPP1CA LOC388720 MYO15A PILRA HSP90AB2P ATP6V1D RPL26L1 PTPRCACTBL3 SNX9 MPP6 RAN LOC442497 PCYOX1 GNG2 RDX A26C1A ANKFY1 TMED9 SDCBPHIST2H4B UFC1 DOCK10 STX3 hCG_1757335 FAM49B C3orf10 STXBP1 HLA-A29.1CUTA MYO1G STXBP2 LOC653269 ATP6V1H FLJ21438 TPI1 A26C1B VPS24 SLC38A1EZR LOC100128936 CMPK1 FERMT3 YWHAE LOC100130553 UPB1 ITFG3 TUBA1ALOC100133382 CLIC5 HIST1H2AH WDR1 LOC100133739 MUPCDH SLAMF6 PDCD6IPAP2A2 CLIC6 TMC8 GPA33 ALDH3B1 SIAE LOC153364 TUBA1B FASLG CPVL SVIPTUBB2C ATP4A RHOF TMEM189- UBE2V1 CAPN7 CAPS ARL15 hCG_16001 DDAH1COL12A1 ZNHIT6 FABP5L7 PGLS DMBT1 GIPC2 Del(X)1Brd SAMM50 DSP PCDH24ABP1 CLIC4 EGFR VPS13C ACTN3 CHMP2B EPHA5 CC2D1A AFM ULK3 EPHB1 EPS8L1AKT1 RNF11 FAT C10orf18 ALDH3A2 VPS4A HSD17B4 CHCHD3 ALOX12P2 ARFIP1L1CAM C2orf18 ANXA2P1 CHMP2A LAMA5 C17orf80 KRT33B SMPDL3B MUC4 EPN3MYOC PACSIN3 NOTCH1 UACA SERPINE1 EHD4 PPP2R1B VPS13D PIK3CA EHD3 PTPRFAPPL2 NRP1 HEBP1 SORT1 ARL8B SPRY1 VPS28 SERPINB3 DDX19A EMILIN1 DCXRSELP NAGK LRG1 RHCG FSCN1 ITLN1 AZGP1P1 CHMP5 TGFB1 CCDC132 LOC728533VTA1 CLTCL1 OTUB1 ALDH7A1 RAB14 CHST1 CDK5RAP2 AXL GPRC5B EIF3I MBD5 CFBCAB39 TNFSF10 SLC22A11 C1S RAB8B MAP7 SUSD2 CAT TM7SF3 COPB2 SUCNR1 CD47MXRA8 HEPH BDH2 CD151 C11orf59 NIT2 CDH13 MOBKL1B CIB1 RPL23AP13 CFTRUEVLD SLC34A2 FAM20C CEACAM8 TSNAXIP1 SLC6A14 SLC12A9 AP1S1 GPRC5C DIP2ARAB25 CLTA GNG12 TNP03 SMURF 1 CNGB1 BAIAP2L1 FER1L3 TMEM27 COL1A1 MUC13CNTLN RAB22A COL1A2 CHMP1B TUBB4Q NDRG3 COL2A1 SLC44A2 KIF15 ERMN COL3A1CPNE5 SERINC1 TAOK1 COL4A1 TMBIM1 PDIA2 KIAA1529 COL4A2 EPS8L3 EP S 8L2RNF213 COL4A3 MMRN2 PLVAP WIZ COL5A1 TTYH3 MYADM ACE2 COL5A2 SLC44A4MUC16 PLEKHAl C0L7A1 RAB1B KRT25 SCPEP1 COMP RAB33B SERINC5 AASDHPPTCPS1 RBP5 LOC440264 FIGNL1 CSF1 C5orf32 AGT PBLD VCAN ABHD14B ALPP KIF9SLC25A10 MOBKL1A APOA2 LEPRE1 CTBP2 ARRDC1 APOB RAB17 CTNNA2 APOE IKZF5DCTN1 FAM125A SERPING1 MMP25 DECR1 SNX18 ClQB MPP5 DNASE1Ll CHMP4B C1RTEKT3 ENG MITD1 C4A ALDH8A1 STX2 S100A16 C4B SLC13A3 ETFB CPNE8 C4BPADUSP26 F2R Clorf58 C4BPB GGCT F8 GLIPR2 CD5L TMEM38A ACSL1 TUBB FCN1Clorf116 FAP ATP6V1C2 FCN2 GDPD3 FBLN1 FTLL1 FGB OR2A4 FBN1 PEF1 FGGFAM65A FBN2 SERPINA3 GRIN1 NARG1L FEN1 ACP2 MSH6 CHMP6 FLT1 ACPP HBA1DYNC2H1 FUCA2 ACTA2 HBA2 PRKRIP1 GAS6 ACTC1 ITGA2B GSTCD GDI1 ACTG2PPARG PIP4K2C GLDC ACY1 PDLIM7 CYBRD1 GNAL APCS CD274 FUZ GRM2 APOD AlBGARMC9 GRM3 APRT ACAT1 NAT13 GRM7 AQP1 AC01 COASY GSTM1 AQP2 ADCY1 UBXN6GSTM5 ARF1 ADFP COL18A1 H2AFX ARF3 ADH5 BHLHB9 HBE1 ARF4 ADH6 WNT5BHMGCS2 ARF5 PARP4 CAB39L TNC ARF6 AHSG ITM2C IDH3B RHOA AK1 L0081691IFRD1 ARL3 ALAD AMN ITGA5 ASAH1 ALCAM SH3BGRL3 ITGB5 ASS1 ALDH2 C9orf58ITPR2 FXYD2 ALDH9A1 BCL2L12 KRT84 BHMT ALDOC RAB34 LAMB1 BST2 ALKTBC1D10A LCN1 C3 AL0X12 GPR98 LGALS8 CA2 ALPL HDHD2 LMNA CA4 ANXA13 ARL6LOXL2 CALB1 A0X1 IQCG LTBP2 CALR APAF1 C2orf16 MAP1A CD9 AP0A4 PARD6BMAT1A CD59 SHROOM2 TXNDC17 MC1R HSPA5 RHOB ABCC11 MCC HSPA6 ARHGAP1FAM40A ME1 HSP90AB1 ARHGDIB SCIN MECP2 HSPD1 ARSE SCRN2 MAP3K1 IDH1 ARSFZNF486 MFAP4 KNG1 ASL ACY3 SCGB2A1 KRAS ASNA1 C11orf52 ALDH6A1 LAMP1ATIC CRB3 MOS LGALS3BP ATP6V1A C20orf114 CITED1 LRP2 ATP6V1B1 NAPRT1NEFH MAN1A1 ATP6V1B2 RG9MTD2 OPRM1 RAB8A ATP6VOC SAT2 OTC MIF ATP6V1C1KIF12 OXTR MME ATP6V1E1 MAL2 PAPPA MUC1 ATP6V0A1 OSBPL1A PC MYH9 ATP6AP1VASN PCOLCE NAGLU AZU1 SLC22Al2 PDGFRB NONO BCR ACSM1 PFKFB3 NPM1 BGNTTC18 PGAM2 NRAS BLMH GSTO2 SERPINE2 P2RX4 BLVRA CLRN3 PLP2 P4HB BLVRBLRRK2 PPP1CC PEBP1 BPI Cl2orf59 SRGN SERPINA5 BTG1 LOC124220 MAP2K6 PFN1BTN1A1 SLC5A10 PSMB7 PFN2 TSPO CCDC105 PSMB10 ABCB1 C1QC Clorf93 PTK7SERPINA1 CAPN5 ARL8A PTPRK PIGR C5 L0C128192 PZP PIK3C2B C9 GALM RAD21PKD1 PTTGlIP LRRC15 RASA1 PLSCR1 CACNA2D1 LOC131691 RDH5 PODXL CALML3H1F00 RPL18 CTSA CAMK4 ENPP6 RPL29 PPIA CAMP CMBL RPS10 PSAP CAPG MUM1L1RPS24 PSMB3 CAPN1 C20orf117 S100A13 PTBP1 CAPN2 SIRPA SAA4 PTPRJ CAPZA2PLEKHA7 ATXN1 RABlA CD14 A2ML1 CLEC11A RAB2A CD80 Cl6orf89 SDC2 RAB3BCD36 T0M1L2 SMARCA4 RAB5A SCARB2 KIF18B SPOCK1 RAB5B CD40 C19orf18 STAT1RAB13 CDC2 PM20D1 STC1 RAB27B CEL PROM2 SURF4 RAB5C CETP GPR155 SYT1RAC1 CTSC SLC36A2 TAGLN RALB AP2M1 VPS37D TCN1 RAP1B CSN1S1 SLC5Al2TERF1 RBM3 CSN2 SLC5A8 TGFB2 RNASE2 CSN3 EML5 TSPAN4 S100A6 ACSL3TBC1D21 TSN S100A11 FOLR1 ZNF114 TSNAX Sl00P B4GALT1 ANO6 C0L14A1 SLC1A1GNAQ SLC5A9 WNT5A SLC2A5 HBB CRTC2 ZNF134 SLC12A1 HBD C20orf106 PXDNSLC12A3 CFH TMEM192 SMC1A SNCG HLA-G ARMC3 OFD1 SNRPD1 HP NAPEPLD COPS3SOD1 HPR C10orf30 STC2 SRI IGHAl ATP6V0D2 ADAM9 TF IGJ STXBP4 CREG1THBS1 IGLC1 C17orf61 CDK5R2 THY1 IGLC2 TXNDC TNF5F18 TMPRSS2 IGLC3LRRC57 MPZL1 TSG101 LAMC1 H5PA12A SEMA5A TUBB2A LPA MAGI3 CLDN1 UBE2NLPL Cllorf47 RGN UMOD LRP1 SLC39A5 5LC16A3 UPK2 LTF Cl2orf51 ARH GEF 1VTN TACSTD2 5LC46A3 LRRFIP2 EIF4H MBL2 VMO1 TAAR2 YWHAB MYH8 5LC26A11CRIPT YWHAG NEB LOC284422 ENTPD4 YWHAZ PON1 CRB2 IFT140 NPHS2 PKN2HIST2H2AB RNF40 RAB7A PROS 1 FAM151A RB1CC1 PSCA MASP1 5LC6A19 PSMD6CUBN RELN PKD1L3 MRC2 BBOX1 PTX3 L0C342897 HDAC5 RAB11A RARS EGFL11RASA4 NAPA SILV SERINC2 SLC25A13 PROM1 THBS2 PDDC1 PSMD14 FCGBP TLR2SLC04C1 TFG CPNE3 TTN SFT2D2 CDIPT MGAM TTR C9orf169 CRTAP GPRC5A TYRP1LOC377711 UNC13B RAB11B VWF OR11L1 ARL6IP5 VAMP3 CLIP2 RAB19 TGOLN2SLC9A3R1 XDH LOC440335 POSTN ITM2B APOL1 HIST2H2BF CLPX NAPSA FCN3LOC441241 TSPAN9 VPS4B SELENBP1 KPRP TMED10 RAB3D SMC3 HSP90AB6P SLC38A3PRDX6 DDX21 LOC643751 IL1RAPL1 KIAA0174 CCPG1 LOC651536 GALNT5 PDCD6ABCG2 LOC652968 PRR4 ARPC4 SFIl AEBP1 ITGAll TSPAN1 MVP AMY1A CLASP2PDZKlIP1 AKAP9 AMY1B EPB41L3 NUTF2 PRG4 AMY1C KIAA0467 FLOT1 AKR1A1AMY2A DULLARD HRSP12 ABCA7 ANGPT1 NOM01 A2M COLEC10 APLP2 KIAA0146 ACP1GNB5 APP SLC39A14 ACTA1 MMRN1 AQP5 DNPEP ACTN4 CLASP1 AZGP1 CA5P14 ACTN1SYNE1 CEACAM1 STX12 ACTN2 NIPBL BMP3 BRMS1 ADAM10 CHRDL2 CA6 ABI3BP AHCYHSPB8 DDR1 PLEKHG3 ALDH1A1 ANGPTL4 CAPNS1 FBXW8 SLC25A4 NIN COL6A2GAPDHS SLC25A5 ZNF571 COPA GREM1 SLC25A6 LRP1B CPD DKK3 ANXA1 CNDP2 DLDSRPX2 ANXA2P2 DNAH7 ETFA IGHV3-11 APOA1 HCN3 GLUD1 IGHV3-7 ARHGDIA EXOC4H5D17B10 IGLV4-3 ARVCF SNX25 IMPDH2 IGLV3-21 TC2N HTATIP2 IGLV1-40HAPLN3 MARVELD2 ST6GALNAC6 ATP1B1 CD163L1 CST4 COPS4 ATP5A1 HRNR CST5HERC5 ATP5B P704P CTSB NUSAP1 ATP51 CD24 DAG1 PLUNC ATP50 COL6A3 DSG2PPME1 B2M COL15A1 TOR1A MBD3 CALM1 COMT ECM1 SLC38A2 CALM2 CP EIF4G1FAM64A CALM3 CPN2 EXT2 GTPBP2 CANX CRABP2 FAT2 DIRAS2 CAPZA1 CRK GP C4DCHS2 CD2 CRYAB FOLH1 QPCTL CD247 CRYM FUT2 PARP16 CD 86 CSElL FUT3TMEM51 CD37 CSK FUT6 MCM10 CD44 CSTB FUT8 CHST12 CD53 CTH GLRX LYARCDC42 CTNS GPCl ODZ3 CDH1 CT SD GPX3 WDR52 CFL1 CTSG IGHA2 ASH1L CFL2DDB1 IGHVa UNC45A COX4I1 DDC IGLa SLC7A10 COX5B DDX3X IVL PNO1 CLDN3DDX5 KRT12 CD248 CSPG4 CFD LAMA4 AHRR CSRP1 DNM2 LAMB2 ZBTB4 CST3 DPYSLGALS7 SPTBN4 CTNNA1 DSC2 LMAN1 LGR6 CTNNB1 DSG3 LPO RNF123 NQ01 ECE1LTBP3 PRDM16 DYNC1H1 MEGF8 DNAJB 9 PARVG EEF1A2 ELA2 MEST RMND5A EFNB1SERPINB1 MGAT1 FAT4 CTTN EPHX2 MGP F1113197 EPHB4 FBL MUC5AC TREML2ERBB2 EVPL MUC7 SVEP1 F5 Fll NEU1 OBFC1 FASN FABP1 NUCB1 ZNF614 FKBP1AACSL4 NUCB2 F1122184 FLNA FAH FURIN DBF4B FLNB EFEMP1 PAM CD276 G6PDFBP1 PLG CMIP GCNT2 FKBP4 FXYD3 ADAMTS12 PDIA3 FKBP5 PLOD2 SPACA1 GSNFRK PLTP VANGL1 HADHA FTH1 PON3 SPRY4 HLA-DMB FUCA1 PPP 1CB HYI HLA-EGABRB2 PRELP FAM108A1 HNRNPA2B1 GALK1 DNAJC3 TMEM47 HNRNPH2 GBE1 HTRA1MYCBPAP HSPAlL GDF2 RARRES1 RAB6C HSPA2 GFRA1 SAA1 FAM71F1 HSPA4 GK2SAA2 ZNF503 HSPA7 GLO1 SEPP 1 PARP10 HSPA9 GLUL SFRP1 SHANK3 HSP9OAA4PGM2A ST3GAL1 LACRT HSP9OAA2 GNG5 SLC5A5 TRIM41 HSP90AB3P GOT1 SLC9A1OXNAD1 HSPE1 GPD1 SLC20A2 LDHAL6B HSPG2 GPM6A SLPI L0C92755 ICAM1 GPTSRPR CACNA2D4 ITGA6 GPX4 STAU1 ARHGAP18 ITGA2 GRB2 HSPA13 AHNAK2 ITGAVGRID1 TGFBI RPLPOP2 GSR TGM1 PGLYRP2 ITGB2 GS S TGM3 RAB39B ITGB4 GS TM2YES1 GYLTL1B JUP HGD HIST2H2AA3 KRT74 CD82 HINT1 HIST2H2BE SLAIN1 KPNB1HNMT GALNT4 LOC122589 KRT2 HNRNPL B4GALT3 NLRP8 KRT5 HPD TNFSF13 PODNKRT8 HPX TNFSF12 C5orf24 KRT13 HRG ANGPTL1 CD109 KRT14 DNAJA1 GCNT3TRIM40 KRT15 HSPB1 TM9SF2 GPR112 KRT16 DNAJB1 DDX23 KRT72 KRT18 CFIADAMTS 3 VTI1A KRT19 IGF2R GPR64 SYT9 LAMP2 IGFALS LHFPL2 KRT80 LGALS4IL1RN ST3GAL6 CCDC64B LYZ IRF6 PRDX4 ATP8B3 ITGA1 MAN1A2 Clorf84 MFGE8EIF6 OS9 LOC149501 MMP7 ITGB8 MGAT4A LOC150786 MYH10 ITIH4 TWF2 WDR49MYL6 KHK CLCA4 NEK10 MY01C KIFC3 TXNDC4 STOML3 MY01D KLK1 PLCB1 SASS6NME1 LBP CES3 DCLK2 NME2 LCN2 B3GAT3 FREM3 PRDX1 LCP1 TOR1B C9orf91PCBP1 LTA4H IGHV30R16-13 TREML2P CHMPlA BCAM IGLV2-11 CCDC129 SERPINF1MAN2A1 IGLV1-44 PAN3 PHB MDH2 IGKV3D-15 MAMDC2 PPIB MFI2 IGKV4-1 RCOR2PRKAR2A MLLT3 C1GALT1C1 LOC283412 PRKDC MLLT4 RACGAP1 LOC283523 PSMA2MNDA EFEMP 2 NOM02 QSOX1 MPO DUOX2 SEC14L4 PYGB MPST SDF4 LCN1L1 RAB6AMY01B CYB5R1 LOC286444 RALA MSRA ERAP 1 TAS2R60 RAP1A MTAP NUDT9KRT18P19 RPL6 MTHFD1 FAM3B LOC343184 RPL8 MYH3 FAM20A LOC345041 RPLP1MY05B FAM55D GNAT3 RPLP2 MY06 ANO1 POLN RPN1 NID1 LRRC16A LOC376693 RPS3NKX6-1 TTC17 ARMS2 RPS7 NQ02 PDGFC LOC387867 RPS13 NP PCDHGB5 LOC388339RPS14 NPC1 CCL28 FLG2 RPS15A NPHS1 UGCGL1 LOC388707 RPS18 NRF1 SEMA3GLOC389141 RPS20 NT5E CORO1B LOC390183 RPS21 PAFAH1B1 NDRG2 KRT8P9 RPS27APAFAH1B2 KIAA1324 LOC391777 RRAS PCBD1 TXNDC16 LOC391833 S100A10 PCK1ARH GAP 23 LOC399942 SDC1 PDCD2 MUTED LOC400389 SDC4 PDE8A TINAGL1LOC400578 SLC1A5 ENPP3 TOR3A LOC400750 SLC2A1 SLC26A4 VWA1 LOC400963PDZK1 CHID1 F1121767 SLC12A2 PEPD TMEM109 LOC401817 SLC16A1 PFKL GAL3ST4 NOM03 SPTBN1 PGD THSD4 LOC439953 SSBP1 PGM1 UXS1 RPL12P6 SSR4 SLC25A3TXND C5 LOC440589 TBCA SERPINA4 CRISPLD1 LOC440917 TCEB1 SERPINB6 LOXL4LOC440991 TFRC SERPINB13 GNPTG LOC441876 TKT PIK3C2A SCGB3A1 LOC442308TSPAN8 PIP CHST14 DIPAS TPM1 PKD2 C1QTNF1 LOC643300 HSP90B1 PKLR C1QTNF3LOC643358 TUBA4A PKHD1 SLC26A9 LOC643531 TUFM PLCD1 FAM129A RPSAP8 TXNPLOD1 HIST2H3C LOC644464 UBA52 PLS1 TPRG1L LOC644745 UBB UBL3 TMPRSS11BLOC645018 UBC PPL C20orf70 LOC645548 UBA1 PPP1R7 PPM1L LOC646127 UBE2V2PRCP GBP6 LOC646316 UGDH PRKCA KRT78 LOC646359 UQCRC2 PRKCD SLC37A2LOC646785 VCP PRKCH NPNT LOC646875 VIL1 PRKCI KRT73 LOC646949 YWHAHPRKCZ HIST2H3A LOC647000 CXCR4 PRNP VWA2 LOC647285 SLC7A5 PRSS8 GSTK1LOC650405 HIST1H4I PRTN3 SBSN LOC650901 HIST1H4A PSMA1 C5 orf46LOC652493 HIST1H4D PSMA3 LRRC26 LOC652797 HIST1H4F PSMA4 C4orf40LOC653162 HIST1H4K PSMA5 L0C440786 PPIAL3 HIST1H4J PSMB1 SCFV LOC653232HIST1H4C PSMB2 LGALS7B HSPBL2 HIST1H4H PSMB5 HIST2H3D LOC728002 HIST1H4BPSMB6 ACAT2 LOC728088 HIST1H4E PSMC5 ACTL6A LOC728576 HIST1H4L PSMD12ADK LOC728590 HIST2H4A PSME2 ANXA8L2 LOC728791 TAGLN2 PTPN6 LOC728979RUVBL1 PTPN13 ANG VAMP8 PTPRO BDNF SNAP23 QDPR CAV1 CALU IQGAP1 RAB27ACD70 CCR4 KRT75 RAP1GDS1 CS CCR5 TJP2 RBL2 DARS C5F2 ROCK2 RBP4 DHX9C5F3 ARPC3 RENBP DPYSL2 DCN ACTR3 RFC1 EEF1D EPO LRPPRC RHEB EPRS F3TRAP1 RNH1 FDPS GPC5 TUBB4 RNPEP FLNC GDF1 GNB2L1 ROB02 XRCC6 GDF9BAIAP2 RP2 GFPT1 GFRA3 HYOU1 RP511 HIST1H1B GRN AGR2 RREB1 HIST1H2BBCXCL2 OLFM4 RYR1 H3F3A GZMA CCT2 S100A4 H3F3B HIST1H2BD ATP5L S100A8HNRNPF HGF CCT8 S100A9 HNRNPK IFNG SLC12A7 SERPINB4 IARS IGFBP3 MASP2SCN10A LAMA3 IGFBP4 IQGAP2 SEC13 LAMB3 IGFBP6 RAB10 SECTM1 LAMC2 IGFBP7PRDX3 SH3BGRL LGAL51 IL1RAP EHD1 5HMT1 NBR1 IL3 TMED2 SHMT2 MARS IL5LMAN2 SLC3A1 MX1 IL6ST YWHAQ SLC4A1 PFKP IL7 GCN1L1 SLC5A1 PLAU IL8RAB35 SLC5A2 PSMB4 IL10 DSTN SLC6A13 PSMC2 IL11 UPK1A SLC9A3 PSMC4 IL13PHB2 SLC15A2 PSMD2 IL15RA RRAS2 SLC25A1 PSMD13 INHBA SEC31A SLC22A2 PYGLINHBB CLSTN1 SLC22A5 RPL10 IP05 PTGR1 SMO RPL15 LIF RAB21 SORD STX4 LRP6CYFIP1 SORL1 TARS LTBP1 SLC44A1 SPAST CLDN5 MMP1 CORO1C SPR TPBG MMP2MTCH2 SPRR3 XPO1 MMP3 QPCT SRC XRCC5 MMP10 PRDX5 ST13 BAT1 NBL1 SND1STK11 HIST1H2BG TNFRSF11B FUR VAMP7 HIST1H2BF OSM LIMA1 SYPL1 HIST1H2BEPDGFA RAB6B SERPINA7 HIST1H2BI PRKCSH KRT20 TECTA HIST1H2BC CCL2 VPS35TGM4 HIST1H4G CCL7 TOMM22 TGFBR3 EIF3A CCL20 AKR1B10 TGM2 EIF3B SFRP45100A14 TLN1 EIF3C 50D3 DIP2B DNAJC7 SLC5A6 SPARC RAP2C UBE2G1HIST2H2AA4 TIMP1 FAM129B UPK1B LOC728358 TIMP2 UGP2 LOC730839 TIMP3AHNAK UPK3A LOC100126583 ICAM5 VPS37B UTRN AARS TNFRSF1A TUBA4B VASP AK2VEGFC ARPC5L VCL APEH GDF5 EPPK1 VDAC1 FAS HIST3H3 ADSL VDAC3 BAXHIST1H2AI AP2A1 XPNPEP2 FMNL1 HIST1H2AL RHOC BTG2 CASP9 HIST1H2AC RHOGGCS1 CD19 HIST1H2AM ASNS BAT2 M54A1 HIST1H2BN PTP4A2 CD22 HIST1H2BM CADDYSF TNFRSF8 HIST1H2BH CBR1 EEA1 SCARB1 HIST1H2B0 CBR3 5TK24 ENTPD1HIST1H3A CCT6A CUL4B CD48 HIST1H3D CDH17 CUL3 CD58 HIST1H3C CEACAM5 ATRNCD74 HIST1H3E COPB1 CDC42BPA CD79B HIST1H3I CLDN4 PPFIA2 CD97 HIST1H3GCLDN7 AKR7A2 41889 HIST1H3J CRYZ PPAP2A CR2 HIST1H3H CD55 ABCB11 CSNK2BHIST1H3B EEF1G MAP2K1IP1 DBI FADD EPHA2 EIF3H DHCR7 IL1RL2 EIF4A1 SLC4A4DLG1 FGF18 EIF4A2 SNX3 DOCK2 FGF16 ENO2 MYH13 DUT HIST1H3F SLC29A1 NAPGECH1 HIST1H2AG EPHB2 FBP2 VAPA HIST1H2BJ EPHB3 SCEL H2AFY NRG2 ESDSUCLA2 PDIA4 GDF3 F7 GGH EIF4A3 FGF19 FLOT2 PROZ ACTR1B GDF11 GARSSQSTM1 OPTN FST GMDS AP1M1 NAMPT LASS1 GNB3 RAB7L1 MPZL2 HPSE HIST1H2AEWASL STIP1 ESM1 HLA-C PLOD3 PKP3 DKK1 HLA-H PGLYRP1 POFUT2 IL17B HPCAL1KALRN QPRT IL19 CLIC3 WBP2 TNFRSF12A IGHα BAZ1B ERO1L IL23A IGHG1 SPAG9H2AFY2 FGFRL1 IGHG2 SLC13A2 RCC2 TREM1 IGHG3 ATP6V0D1 RTN4 IL1F9 IGHG4HGS GLT25D1 CXCL16 IGHM AP4M1 RNASE7 IL22RA1 IGKC ATP6V1F FCRLAHIST1H2BK ITGA3 PTER H2AFV HIST3H2BB KRT3 TRIP10 MRLC2 LOC440093 KRT4SLC9A3R2 PAGE2 PGAM4 KRT6A SLIT2 HIST1H2BA PC-3 KRT6B SLC22A6 SNX33LOC729500 KRT7 KL PTRF KRT18P26 KRT17 KIF3B HIST2H2BC S100A11P RPSASLC22A8 ANXA8 LOC729679 LFNG GRHPR NME1-NME2 KRT17P3 LGALS3 SLC22A13EIF2S1 RCTPI1 LRP4 TMPRSS11D EIF2S3 LOC729903 CD46 GSTO1 EIF4ERP11-556K13.1 MICA NPEPPS EPB41L2 LOC100129982 MYH11 TMEM59 EVI2BLOC100130100 NARS ATP6V1G1 FCER2 LOC100130446 NEDD4 CDC42BPB FGRLOC100130562 RPL10A CREB5 FH LOC100130624 PCNA CROCC GART LOC100130711PLEC1 DHX34 GOT2 LOC100130819 PLXNA1 TMEM63A NCKAP1L LOC100131713PPP2R1A SLK HLA-DPB1 LOC100131863 PSMC6 RUSC2 HLA-DQA1 LOC100132795PSMD3 OXSR1 HNRNPA1 LOC100133211 PSMD11 SLC23A1 HNRNPC LOC100133690 RAC3DOPEY2 HPRT1 SET RAP2A ABI1 ICAM3 CCT6B RAP2B GNPDA1 INSR ACTR3B RPL12TOM1 EIF3E PSMA8 RPLP0 ABCB6 ITGAL ARP11 RPS4X ABCC9 ITGB3 BCHE RPS4Y1HUWEl ITGB7 H2AFZ RPS8 ARPC5 IT1H2 SNRPE RPS16 ACTR2 STMN1 TFPI SPTAN1TSPAN3 LCK ADAMTS1 VAMP1 ARPC2 LSP1 GDF15

TABLE 3 Polypeptide payloads and receivers Ankyrin repeat proteinsFibronectins Lyases General Classes Antibodies Complement receptorsGPI-linked Nanobodies polypeptides Aptamers Cyclic peptides HEAT repeatproteins Nucleic Acids ARM repeat DARPins Hydrolases Polypeptidesproteins Carbohydrates DNAses Kinases Single-chain variable fragments(scFv) Cell surface Enzymes Lipoproteins Tetratricopeptide receptorsrepeat proteins Complement C1 inhibitor C4 binding protein CR3 Factor IC3 Beta chain CD59 CR4 Homologous Receptor restriction factor C3aR CR1Decay-accelerating Membrane cofactor factor (DAF) protein (MCP) C3eR CR2Factor H PRELP Enzymes triacylglycerol bile-acid-CoA hydrolase feruloylesterase phosphatidate lipase phosphatase (S)- bis(2- formyl-CoAphosphatidylglycero methylmalonyl- ethylhexyl)phthalate hydrolasephosphatase CoA hydrolase esterase [acyl-carrier- bisphosphoglyceratefructose- phosphatidylinositol protein] phosphatase bisphosphatasedeacylase phosphodiesterase [phosphorylase] Carboxylic-Esterfumarylacetoacetase phosphodiesterase I phosphatase Hydrolases1,4-lactonase carboxymethylenebuten fusarinine-C phosphoglycerateolidase ornithinesterase phosphatase 11-cis-retinyl-cellulose-polysulfatase galactolipase phosphoglycolate palmitatephosphatase hydrolase 1-alkyl-2- cephalosporin-C gluconolactonasephosphoinositide acetylglycerophosp deacetylase phospholipase Chocholine esterase 2′- cerebroside-sulfatase glucose-1- phospholipase Alhydroxybiphenyl- phosphatase 2-sulfinate desulfinase 2-pyrone-4,6-cetraxate benzylesterase glucose-6- phospholipase A2 dicarboxylatephosphatase lactonase 3′, 5′-bisphosphate chlorogenate hydrolaseglutathione phospholipase C nucleotidase thiolesterase 3- chlorophyllaseglycerol-1- phospholipase D hydroxyisobutyryl- phosphatase CoA hydrolase3′-nucleotidase cholinesterase glycerol-2- phosphonoacetaldehydephosphatase hydrolase 3-oxoadipate enol- choline-sulfataseglycerophosphocholine phosphonoacetate lactonase phosphodiesterasehydrolase 3-phytase choloyl-CoA hydrolase Glycosidases, i.e.phosphonopyruvate enzymes that hydrolase hydrolyse O- and S- glycosylcompounds 4-hydroxybenzoyl- chondro-4-sulfatase glycosulfatasephosphoprotein CoA thioesterase phosphatase 4- chondro-6-sulfataseGlycosylases Phosphoric-diester methyloxaloacetate hydrolases esterase4-phytase citrate-lyase deacetylase histidinol- Phosphoric-monoesterphosphatase hydrolases 4- cocaine esterase hormone-sensitivePhosphoric-triester pyridoxolactonase lipase hydrolases 5′-nucleotidasecutinase Hydrolysing N- phosphoserine glycosyl compounds phosphatase6-acetylglucose cyclamate Hydrolysing S- poly(3- deacetylasesulfohydrolase glycosyl compounds hydroxybutyrate) depolymerase 6-Cysteine endopeptidases hydroxyacylglutathione poly(3-phosphogluconolactonase hydrolase hydroxyoctanoate) depolymerasea-amino-acid Cysteine-type hydroxybutyrate- polyneuridine- esterasecarboxypeptidases dimer hydrolase aldehyde esterase a-Amino-acyl-D-arabinonolactonase hydroxymethylglutaryl- protein-glutamate peptidehydrolases CoA hydrolase methylesterase acetoacetyl-CoA deoxylimonateA-ring- iduronate-2-sulfatase quorum-quenching hydrolase lactonaseN-acyl-homoserine lactonase acetoxybutynylbithiophene dGTPaseinositol-phosphate retinyl-palmitate phosphatase esterase deacetylaseacetylajmaline dihydrocoumarin juvenile-hormone Serine dehyrdataseesterase hydrolase esterase or serine hydroxymethyl transferaseacetylalkylglycerol Dipeptidases kynureninase Serine acetylhydrolaseendopeptidases acetylcholinesterase Dipeptide hydrolasesL-arabinonolactonase serine- ethanolaminephosphate phosphodiesteraseacetyl-CoA Dipeptidyl-peptidases limonin-D-ring- Serine-type hydrolaseand tripeptidyl- lactonase carboxypeptidases peptidases acetylesteraseDiphosphoric-monoester lipoprotein lipase S-formylglutathione hydrolaseshydrolase acetylpyruvate disulfoglucosamine-6- L-rhamnono-1,4- sialateO- hydrolase sulfatase lactonase acetylesterase acetylsalicylatedodecanoy[acyl- lysophospholipase sinapine esterase deacetylasecarrier-protein hydrolase acetylxylan Endodeoxyribonucleases mannitol-1-Site specific esterase producing 3′- phosphatase endodeoxyribonucleases:phosphomonoesters cleavage is not sequence specific acid phosphataseEndodeoxyribonuclease Metallocarboxypeptidases Site-specific producing5′- endodeoxyribonucle phosphomonoesters that are specific for alteredbases. Acting on acid Endopeptidases of Metalloendopeptidases.Site-specific anhydrides to unknown catalytic endodeoxyribonucleases:catalyse mechanism cleavage is transmembrane sequence specific movementof substances Acting on acid Endoribonucleasesmethylphosphothioglycerate sphingomyelin anhydrides to producing 3′-phosphatase phosphodiesterase facilitate cellular phosphomonoesters andsubcellular movement Acting on GTP to Endoribonucleasesmethylumbelliferyl- S- facilitate cellular producing 5′- acetatedeacetylase succinylglutathione and subcellular phosphomonoestershydrolase movement Acting on Endoribonucleases that monoterpenee-lactone steroid-lactonase phosphorus- are active with either hydrolasenitrogen bonds ribo- or deoxyribonucleic acids and produce 3′-phosphomonoesters Acting on sulfur- Endoribonucleases that N- sterolesterase nitrogen bonds are active with either acetylgalactosamine-ribo- or 4-sulfatase deoxyribonucleic acids and produce 5′-phosphomonoesters actinomycin Enzymes acting on acid N- steryl-sulfataselactonase anhydrides acetylgalactosamine- 6-sulfatase acylcarnitineEnzymes Acting on N- succinyl-CoA hydrolase carbon-carbon bondsacetylgalactosaminoglycan hydrolase deacetylase acyl-CoA Enzymes actingon N-acetylglucosamine- sucrose-phosphate hydrolase carbon-nitrogenbonds, 6-sulfatase phosphatase other than peptide bonds acylglycerollipase Enzymes acting on N-sulfoglucosamine sugar-phosphatasecarbon-phosphorus sulfohydrolase bonds acyloxyacyl Enzymes acting onoleoyl-[acyl-carrier- Sulfuric-ester hydrolase carbon-sulfur bondsprotein] hydrolase hydrolases acylpyruvate Enzymes Acting on Omegapeptidases tannase hydrolase ether bonds ADAMTS13 Enzymes acting onorsellinate-depside Thioester hydrolases halide bonds hydrolaseAdenosine Enzymes acting on oxaloacetase Thioether and deaminase peptidebonds trialkylsulfonium (peptidases) hydrolases adenylyl- Enzymes actingon palmitoyl[protein] Threonine [glutamate— phosphorus-nitrogenhydrolase endopeptidases ammonia ligase bonds hydrolase ADP-dependentEnzymes acting on palmitoyl-CoA thymidine medium-chain- sulfur-nitrogenbonds hydrolase phosphorylase acyl-CoA hydrolase ADP-dependent Enzymesacting on pectinesterase trehalose-phosphatase short-chain-acyl-sulfur-sulfur bonds CoA hydrolase ADP- Ether hydrolases. Peptidylpeptide triacetate-lactonase phosphoglycerate hydrolases phosphatasealkaline Exodeoxyribonucleases Peptidyl-amino-acid Triphosphoric-phosphatase producing 5′- hydrolases monoester phosphomonoestershydrolases all-trans-retinyl- Exonucleases that are Peptidylamino-acidtrithionate hydrolase palmitate active with either ribo- hydrolases orhydrolase or deoxyribonucleic acylamino-acid acids and produce 3′-hydrolases phosphomonoesters aminoacyl-tRNA Exonucleases that arePeptidyl-dipeptidases tropinesterase hydrolase active with either ribo-or deoxyribonucleic acids and produce 5′- phosphomonoestersAminopeptidases Exoribonucleases phenylacetyl-CoA ubiquitin producing3′- hydrolase thiolesterase phosphomonoesters arylesteraseExoribonucleases Phenylalanine UDP-sulfoquinovose producing 5′- ammonialyase synthase phosphomonoesters. arylsulfatase Factor IX Phenylalanineuricase hydroxylase Asparaginase Factor VIII pheophorbidaseuronolactonase Aspartic fatty-acyl-ethyl-ester phloretin hydrolasewax-ester hydrolase endopeptidases synthase b-diketone hydrolasephorbol-diester xylono-1,4-lactonase hydrolase

TABLE 4 Targets General Classes of Targets Microbes Polypeptides DNAAmino Acids Fungi Toxins RNA Prions Bacteria Lipids Parasites CytokinesVirus Cells Cellular Debris Infectious Disease-Related TargetsLipopolysaccharides Cell invasion protein Intermedilysin Secretedeffector protein sptP Zona occludens Cholera enterotoxin Invasionprotein Seeligeriolysin toxin sipA Actin Cysteine protease Iota toxinSerine protease polymerization component Ia protein RickA ActinCytolethal distending Ivanolysin Shiga toxin polymerization toxinprotein RickA Adenosine Cytolysin LepB Sphingomy elinase monophosphate-protein transferase vopS adenylate cyclase Cytotoxic necrotizing Lethalfactor Staphylokinase factor Adenylate cyclase Cytotoxin LeukotoxinStreptokinase ExoY ADP- Dermonecrotic toxin Listeriolysin Streptolysinribosyltransferase enzymatic component Aerolysin DeubiquitinaseMicrobial Streptopain collagenase Alpha-toxin Diphtheria toxin Outermembrane Suilysin protein IcsA autotransporter AlveolysinEnterohemolysin Panton-Valentine Superantigen Leucocidin F AlveolysinEnterotoxin Perfringolysin T3SS secreted effector EspF Anthrolysin OEpidermal cell Pertussis toxin Tetanus toxin differentiation inhibitorArp2/3 complex- Exoenzyme Phospholipase Tir activating protein rickABinary ADP- Exotoxin Plasminogen TolC ribosyltransferase activator CDTtoxin Botulinum G-nucleotide exchange Pneumolysin Toxic shock neurotoxinfactor syndrome toxin C2 toxin, Guanine nucleotide Protective antigenZink- component II exchange factor sopE carboxypeptidase CagA Heatstable enterotoxin Protein kinase Zink- carboxypeptidase Calmodulin-IgA-specific serine Pyolysin Zn-dependent sensitive adenylateendopeptidase peptidase cyclase autotransporter Cell cycle Inositolphosphate RTX toxin inhibiting factor phosphatase sopB Lipid & CellTargets Circulating tumor very low density lipid Triglycerides Fattyacids cells (VLDL) Metastases high density lipoprotein ChylomicronsCholesterol Eukaryotic cells low density lipoprotein Apolipoproteins

TABLE 5 Cancers Acute Colorectal cancer Macroglobulinemia,Pleuropulmonary lymphoblastic Waldenstrom Blastoma, leukaemia (ALL)Childhood Acute myeloid Craniopharyngioma, Male Breast Cancer Pregnancyand leukaemia (AML) Childhood Breast Cancer Adrenocortical CutaneousT-Cell Malignant Fibrous Primary Central Carcinoma Lymphoma Histiocytomaof Bone Nervous System and Osteosarcoma (CNS) Lymphoma AIDS-RelatedDuctal Carcinoma In Melanoma Prostate Cancer Kaposi Sarcoma Situ (DCIS)AIDS-Related Embryonal Tumors, Merkel Cell Carcinoma Rare cancerslymphoma Childhood Anal Cancer Endometrial Cancer Mesothelioma RectalCancer Appendix Cancer Ependymoma, Metastatic Squamous Renal cellChildhood Neck Cancer with carcinoma Occult Primary Astrocytomas,Epithelial cancer Midline Tract Renal Pelvis and Childhood CarcinomaUreter, Transitional Involving NUT Gene Cell Cancer Atypical EsophagealCancer Molar pregnancy Retinoblastoma Teratoid/Rhabdoid Tumor, ChildhoodBasal Cell Esthesioneuroblastoma, Mouth and Rhabdomyosarcoma CarcinomaChildhood oropharyngeal cancer Bile duct cancer Ewing sarcoma MultipleEndocrine Salivary Gland Neoplasia Syndromes, Cancer Childhood Bladdercancer Extragonadal Germ Multiple Sarcoma Cell Tumor Myeloma/Plasma CellNeoplasm Bone cancer Extrahepatic Bile Duct Mycosis Fungoides Secondarycancers Cancer Bowel cancer Eye Cancer Myelodysplastic Sézary SyndromeSyndromes Brain Stem Gallbladder Cancer Myelodysplastic/Myelo SkinCancer Glioma, Childhood proliferative Neoplasms Brain tumours Gastriccancer Myeloproliferative Skin cancer (non Disorders, Chronic melanoma)Breast cancer Gastrointestinal Nasal Cavity and Small Cell LungCarcinoid Tumor Paranasal Sinus Cancer Cancer Bronchial Tumors, GermCell Tumor Nasopharyngeal cancer Small Intestine Childhood CancerBurkitt Lymphoma Gestational Neuroblastoma Soft Tissue trophoblastictumours Sarcoma (GTT) Cancer of Glioma Non-Hodgkin Squamous Cell unknownprimary Lymphoma Carcinoma Cancer spread to Hairy cell leukaemiaNon-Small Cell Lung Squamous Neck bone Cancer Cancer with OccultPrimary, Metastatic Cancer spread to Head and neck cancer Oesophagealcancer Stomach (Gastric) brain Cancer Cancer spread to Heart Cancer,Oral Cancer Stomach cancer liver Childhood Cancer spread toHepatocellular (Liver) Oral Cavity Cancer T-Cell Lymphoma, lung CancerCutaneous - see Mycosis Fungoides and Sézary Syndrome Carcinoid TumorHistiocytosis, Oropharyngeal Cancer Testicular cancer Langerhans CellCarcinoma of Hodgkin Lymphoma Osteosarcoma (Bone Throat Cancer UnknownPrimary Cancer) Cardiac (Heart) Hypopharyngeal Osteosarcoma and Thymomaand Tumors, Cancer Malignant Fibrous Thymic Carcinoma ChildhoodHistiocytoma Central Nervous Intraocular Melanoma Ovarian Cancer ThyroidCancer System Atypical Teratoid/Rhabdoid Tumor, Childhood CentralNervous Islet Cell Tumors, Pancreatic Cancer Transitional Cell SystemEmbryonal Pancreatic Cancer of the Renal Tumors, Neuroendocrine Pelvisand Ureter Childhood Tumors Central Nervous Kidney cancer PancreaticUnknown primary System, Neuroendocrine cancer Childhood Tumors (IsletCell Tumors) Cervical cancer Langerhans Cell Papillomatosis, Ureter andRenal Histiocytosis Childhood Pelvis, Transitional Cell Cancer Chordoma,Laryngeal Cancer Paraganglioma Urethral Cancer Childhood ChoriocarcinomaLeukemia Parathyroid Cancer Uterine Cancer, Endometrial Chronic Lip andOral Cavity Penile Cancer Uterine Sarcoma Lymphocytic Cancer Leukemia(CLL) Chronic myeloid Liver cancer Pharyngeal Cancer Vaginal cancerleukaemia (CML) Chronic Lobular Carcinoma In Pheochromocytoma VulvarCancer Myeloproliferative Situ (LCIS) Disorders Colon cancer LowMalignant Pituitary Tumor Waldenstrom Potential Tumor MacroglobulinemiaLymphoma Lung Cancer Plasma Cell WilmsTumor Neoplasm/Multiple Myeloma

The present invention has been described with respect to representativeexamples that are to be considered illustrative embodiments that do notlimit the scope of the invention which is defined solely by the claims.All references to publications, including scientific publications,treatises, textbooks, patent applications and issued patents are herebyincorporated by reference for all purposes.

1. An extracellular vesicle comprising one or more immunomodulatingcomponent(s) that, upon contact with a macrophage, selectivelyrepolarizes the macrophage from an M2 to an M1 phenotype.
 2. Theextracellular vesicle of claim 1, wherein the one or moreimmunomodulating component(s) inhibit(s) at least one macrophage targetgene.
 3. The extracellular vesicle of claim 1 or 2, wherein theextracellular vesicle is an exosome.
 4. The extracellular vesicle of anyone of claims 1 to 3, wherein the immunomodulating component is anucleic acid.
 5. The extracellular vesicle of claim 4, wherein thenucleic acid is an inhibitory RNA.
 6. The extracellular vesicle of claim5, wherein the inhibitory RNA is an antisense RNA, an siRNA, an shRNA, amiRNA, a ncRNA, a pri-miRNA or a pre-miRNA.
 7. The extracellular vesicleof claim 4, wherein the nucleic acid is an antisense oligonucleotide(ASO).
 8. The extracellular vesicle of any one of claims 1-7, whereinthe immunomodulating component inhibits at least one gene is selectedfrom the group consisting of: KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta,Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO,TDO, Arginase, Glutaminase, CEBP/β, Pi3Kγ, and PKM2.
 9. Theextracellular vesicle of claim 8, wherein the at least one gene isselected from the group consisting of: STAT3, STAT6, CEBP/β, Pi3Kγ,KRAS, and HIF1-alpha.
 10. The extracellular vesicle of claim 9, whereinthe immunomodulating component is an antisense oligonucleotidecomprising a sequence at least 95% identical to a sequence selected fromSEQ ID NOs:1-6.
 11. The extracellular vesicle of claim 10, wherein theimmunomodulating component is an antisense oligonucleotide comprising asequence selected from SEQ ID NOs:1-6.
 12. The extracellular vesicle ofclaim 9, wherein the at least one gene is STAT3.
 13. The extracellularvesicle of claim 12, wherein the immunomodulating component is anantisense oligonucleotide that targets STAT3.
 14. The extracellularvesicle of claim 9, wherein the at least one gene is KRAS.
 15. Theextracellular vesicle of claim 14, wherein the immunomodulatingcomponent is an inhibitory RNA that targets wild-type human KRAS. 16.The extracellular vesicle of claim 15, wherein the inhibitory RNA alsotargets mouse Kras^(G12D).
 17. The extracellular vesicle of any one ofclaims 1-16, wherein the macrophage is a tumor resident macrophage. 18.The extracellular vesicle of claim 17, wherein the tumor is a pancreatictumor.
 19. The extracellular vesicle of any one of claims 1-18, furthercomprising an additional immunomodulating component.
 20. Theextracellular vesicle of claim 19, wherein the additionalimmunomodulating component is a small molecule drug, an antibody oractive fragment thereof or a therapeutic protein or active fragmentthereof.
 21. The extracellular vesicle of claim 20, wherein theadditional immunomodulating component is an antibody or active fragmentthereof.
 22. The extracellular vesicle of claim 21, wherein the antibodyor active fragment thereof is an immune checkpoint inhibitor that bindsto CTLA-4, PD-1, or PD-L1 or an inhibitor that binds to CSF1-R.
 23. Theextracellular vesicle of claim 22, wherein the antibody or activefragment thereof comprises CDRs that are at least 95% identical to theCDRs of Ipilimumab, or at least 95% identical to the CDRs of Nivolumab,or at least 95% identical to the CDRs of Cemiplimab, or at least 95%identical to the CDRs of Pembrolizumab, or at least 95% identical to theCDRs of Atezolizumab, or at least 95% identical to the CDRs of Avelumab,or at least 95% identical to the CDRs of Durvalumab, or at least 95%identical to the CDRs of Pexidartinib, or at least 95% identical to theCDRs of PLX7486, or at least 95% identical to the CDRs of ARRY-382, orat least 95% identical to the CDRs of JNJ-40346527, or at least 95%identical to the CDRs of BLZ945, or at least 95% identical to the CDRsof Emactuzumab, or at least 95% identical to the CDRs of AMG820, or atleast 95% identical to the CDRs of IMC-CS4, or at least 95% identical tothe CDRs of Cabiralizumab.
 24. The extracellular vesicle of claim 23,wherein the antibody or active fragment thereof is at least one antibodyselected from the group consisting of Ipilimumab, Nivolumab, Cemiplimab,Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib,PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4and Cabiralizumab.
 25. The extracellular vesicle of claim 22, whereinthe antibody or active fragment thereof is at least one antibody thatcompetes for binding with antibody selected from the group consisting ofIpilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab,Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527,BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab.
 26. Theextracellular vesicle of any one of claims 20-25, further comprisingPTGFRN or a fragment thereof.
 27. The extracellular vesicle of claim 26,wherein the antibody or fragment thereof is fused to the PTGFRN orfragment thereof.
 28. The extracellular vesicle of any one of claims1-27, wherein the comparison is determined using an assay selected fromthe group consisting of an extracellular vesicle uptake assay, a targetgene expression assay, a downstream gene expression assay, a cytokinerelease assay and a macrophage cell surface protein assay.
 29. Theextracellular vesicle of any one of the above claims wherein the M2macrophage is a tumor associated macrophage selected from the groupconsisting of a M2a, M2b, and M2c macrophage.
 30. The extracellularvesicle of any one of the above claims wherein the M1 macrophageexhibits increased secretion of inflammatory cytokines and chemokinesselected from the group consisting of INFγ, IL-12, IL-23, TNFα, IL-6,IL-1, CSCL9, CXCL10 and CXCL11 compared to the M2 macrophage prior topolarization.
 31. The extracellular vesicle of any one of the aboveclaims wherein the M1 macrophage exhibits decreased secretion ofimmunosuppressive cytokines and chemokines selected from the groupconsisting IL-10, TGFβ, PGE2, CCL2, CCL17, CCL18, CCL22 and CCL24compared to the M2 macrophage prior to polarization.
 32. Theextracellular vesicle of any one of the above claims wherein the M1macrophage expresses increased tumor associated antigen compared to theM2 macrophage prior to polarization.
 33. The extracellular vesicle ofany one of the above claims wherein the M1 macrophage increasesstimulation of CD8⁺ T-Cells and/or Natural Killer cells compared to theM2 macrophage prior to polarization.
 34. A pharmaceutical compositioncomprising the extracellular vesicle of any one of claims 1-33.
 35. Amethod of treating a disease in a patient in need thereof comprisingadministering the extracellular vesicle of any one of claims 1-33 or thepharmaceutical composition of claim 34 to the patient, thereby treatingthe disease in the patient.
 36. The method of claim 35, wherein thedisease is a cancer.
 37. The method of claim 35 or 36, wherein thepatient is human.
 38. The method of claim any one of claims 35-37,wherein the immunomodulating component is an inhibitory RNA targeting aproto-oncogene.
 39. The method of claim 38, wherein the proto-oncogeneis human KRAS.
 40. The method of claim 39, wherein the cancer ispancreatic cancer.
 41. The method of any one of claims 35-40, furthercomprising performing at least a second therapy.
 42. The method of claim41, wherein the second therapy comprises a surgical therapy,chemotherapy, radiation therapy, cryotherapy, hormonal therapy, orimmunotherapy.
 43. The method of any one of claims 26-33, wherein theadministering is via a route selected from the group consisting ofintravenous, intraperitoneal and intratumoral administration.
 44. Amethod of modulating gene expression in a macrophage, comprising:contacting the macrophage with an extracellular vesicle comprising oneor more immunomodulating components that inhibit at least one gene andthereby increase macrophage polarization from an M2 to an M1 phenotype,as compared to contacting the macrophage with equimolar amount(s) of theimmunomodulating components alone.
 45. The method of claim 44, whereinthe contacting is ex vivo or in vivo.
 46. The method of claim 45,wherein the contacting is in vivo.
 47. The method of claim 46, whereinthe contacting in vivo comprises administering the extracellular vesicleto a subject.
 48. The method of claim 46, wherein the administering isvia a route selected from the group consisting of intravenous,intraperitoneal and intratumoral administration.
 49. The method of anyone of claims 46-48, wherein the subject is human.
 50. The method of anyone of claims 46-49, wherein the subject is suffering from a conditionselected from cancer and fibrosis.
 51. The method of claim 50, whereinthe condition is pancreatic cancer.
 52. The method of any one of claims44-51, wherein the extracellular vesicle is an exosome.
 53. The methodof any one of claims 44-50, wherein the immunomodulating component is anucleic acid.
 54. The method of claim 53, wherein the nucleic acid is aninhibitory RNA.
 55. The method of claim 54, wherein the inhibitory RNAis an antisense RNA, an siRNA, an shRNA, a miRNA, a ncRNA, a pri-miRNAor a pre-miRNA.
 56. The method of claim 55, wherein the nucleic acid isan antisense oligonucleotide (ASO).
 57. The method of any one of claims44-56, wherein the at least one gene is selected from the groupconsisting of: KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1,AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase,Glutaminase, CEBP/β, Pi3Kγ, and PKM2.
 58. The method of claim 57,wherein the at least one gene is selected from the group consisting of:STAT3, STAT6, CEBP/β, Pi3Kγ, KRAS, and HIF1-alpha.
 59. The method ofclaim 58, wherein the immunomodulating component is an antisenseoligonucleotide comprising a sequence at least 95% identical to asequence selected from SEQ ID NOs:1-6.
 60. The method of claim 59,wherein the immunomodulating component is an antisense oligonucleotidecomprising a sequence selected from SEQ ID NOs:1-6.
 61. The method ofclaim 58, wherein the at least one gene is STAT3.
 62. The method ofclaim 61, wherein the immunomodulating component is an antisenseoligonucleotide that targets STAT3.
 63. The method of claim 58, whereinthe at least one gene is KRAS.
 64. The method of claim 63, wherein theimmunomodulating component is an inhibitory RNA that targets wild-typehuman KRAS.
 65. A method of treating pancreatic cancer in a subjectcomprising: administering to the subject an extracellular vesiclecomprising an inhibitory RNA targeting human wild-type KRAS; wherein thetreatment increases the percentage of polarization of tumor-residentmacrophages from an M2 to an M1 phenotype to a greater level than thatobserved in a patient treated with an inhibitory RNA targeting humanKRAS^(G12D).
 66. The method of claim 65, wherein the percentage ofpolarization of tumor-resident macrophages is determined using anex-vivo assay of tumor-resident macrophages obtained from a tumorsample.